Methods and monitoring of treatment with a wnt pathway inhibitor

ABSTRACT

Methods for treating diseases such as cancer comprising administering a Wnt pathway inhibitor, either alone or in combination with other anti-cancer agents, and monitoring for skeletal-related side effects and/or toxicity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. Provisional ApplicationNo. 61/760,523, filed Feb. 4, 2013, which is hereby incorporated byreference herein in its entirety.

FIELD OF INVENTION

The present invention relates to the field of treating diseases with aWnt pathway inhibitor. More particularly, the invention provides methodsfor treating cancer comprising administering a Wnt pathway inhibitor,either alone or in combination with other anti-cancer agents, andmonitoring for side effects and/or toxicity.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:2293_(—)1060001.ascii.txt; Size: 101 kilobytes; and Date of Creation:Jan. 30, 2014) is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the developed world,with over one million people diagnosed with cancer and 500,000 deathsper year in the United States alone. Overall it is estimated that morethan 1 in 3 people will develop some form of cancer during theirlifetime. There are more than 200 different types of cancer, four ofwhich—breast, lung, colorectal, and prostate—account for almost half ofall new cases (Siegel et al., 2011, CA: A Cancer J. Clin. 61:212-236).

Signaling pathways normally connect extracellular signals to the nucleusleading to expression of genes that directly or indirectly control cellgrowth, differentiation, survival, and death. In a wide variety ofcancers, signaling pathways are dysregulated and may be linked to tumorinitiation and/or progression. Signaling pathways implicated in humanoncogenesis include, but are not limited to, the Wnt pathway, theRas-Raf-MEK-ERK or MAPK pathway, the PI3K-AKT pathway, the CDKN2A/CDK4pathway, the Bc1-2/TP53 pathway, and the Notch pathway.

The Wnt signaling pathway has been identified as a potential target forcancer therapy. The Wnt signaling pathway is one of several criticalregulators of embryonic pattern formation, post-embryonic tissuemaintenance, and stem cell biology. More specifically, Wnt signalingplays an important role in the generation of cell polarity and cell fatespecification including self-renewal by stem cell populations.Unregulated activation of the Wnt pathway is associated with numeroushuman cancers where it is believed the activation can alter thedevelopmental fate of cells. The activation of the Wnt pathway maymaintain tumor cells in an undifferentiated state and/or lead touncontrolled proliferation. Thus carcinogenesis can proceed byovertaking homeostatic mechanisms which control normal development andtissue repair (reviewed in Reya & Clevers, 2005, Nature, 434:843-50;Beachy et al., 2004, Nature, 432:324-31).

The Wnt signaling pathway was first elucidated in the Drosophiladevelopmental mutant wingless (wg) and from the murine proto-oncogeneint-1, now Wnt1 (Nusse & Varmus, 1982, Cell, 31:99-109; Van Ooyen &Nusse, 1984, Cell, 39:233-40; Cabrera et al., 1987, Cell, 50:659-63;Rijsewijk et al., 1987, Cell, 50:649-57). Wnt genes encode secretedlipid-modified glycoproteins of which 19 have been identified inmammals. These secreted ligands activate a receptor complex consistingof a Frizzled (FZD) receptor family member and low-density lipoprotein(LDL) receptor-related protein 5 or 6 (LRP5/6). The FZD receptors areseven transmembrane domain proteins of the G-protein coupled receptor(GPCR) superfamily and contain a large extracellular N-terminal ligandbinding domain with 10 conserved cysteines, known as a cysteine-richdomain (CRD) or Fri domain. There are ten human FZD receptors, FZD1,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD 10. DifferentFZD CRDs have different binding affinities for specific Wnt proteins (Wu& Nusse, 2002, J. Biol. Chem., 277:41762-9), and FZD receptors have beengrouped into those that activate the canonical β-catenin pathway andthose that activate non-canonical pathways (Miller et al., 1999,Oncogene, 18:7860-72).

A role for Wnt signaling in cancer was first uncovered with theidentification of Wnt1 (originally int1) as an oncogene in mammarytumors transformed by the nearby insertion of a murine virus (Nusse &Varmus, 1982, Cell, 31:99-109). Additional evidence for the role of Wntsignaling in breast cancer has since accumulated. For instance,transgenic over-expression of β-catenin in the mammary glands results inhyperplasias and adenocarcinomas (Imbert et al., 2001, J. Cell Biol.,153:555-68; Michaelson & Leder, 2001, Oncogene, 20:5093-9) whereas lossof Wnt signaling disrupts normal mammary gland development (Tepera etal., 2003, J. Cell Sci., 116:1137-49; Hatsell et al., 2003, J. MammaryGland Biol. Neoplasia, 8:145-58). In human breast cancer, β-cateninaccumulation implicates activated Wnt signaling in over 50% ofcarcinomas, and though specific mutations have not been identified,up-regulation of Frizzled receptor expression has been observed (Brennan& Brown, 2004, J. Mammary Gland Biol. Neoplasia, 9:119-31; Malovanovicet al., 2004, Int. J. Oncol., 25:1337-42).0

Activation of the Wnt pathway is also associated with colorectal cancer.Approximately 5-10% of all colorectal cancers are hereditary with one ofthe main forms being familial adenomatous polyposis (FAP), an autosomaldominant disease in which about 80% of affected individuals contain agermline mutation in the adenomatous polyposis coli (APC) gene.Mutations have also been identified in other Wnt pathway componentsincluding Axin and β-catenin. Individual adenomas are clonal outgrowthsof epithelial cells containing a second inactivated allele, and thelarge number of FAP adenomas inevitably results in the development ofadenocarcinomas through additional mutations in oncogenes and/or tumorsuppressor genes. Furthermore, activation of the Wnt signaling pathway,including loss-of-function mutations in APC and stabilizing mutations inβ-catenin, can induce hyperplastic development and tumor growth in mousemodels (Oshima et al., 1997, Cancer Res., 57:1644-9; Harada et al.,1999, EMBO J., 18:5931-42).

Similar to breast cancer and colon cancer, melanoma often hasconstitutive activation of the Wnt pathway, as indicated by the nuclearaccumulation of β-catenin. Activation of the Wnt/β-catenin pathway insome melanoma tumors and cell lines is due to modifications in pathwaycomponents, such as APC, ICAT, LEF1 and β-catenin (see e.g., Lame etal., 2006, Frontiers Biosci., 11:733-742). However, there areconflicting reports in the literature as to the exact role ofWnt/β-catenin signaling in melanoma. For example, one study found thatelevated levels of nuclear β-catenin correlated with improved survivalfrom melanoma, and that activated Wnt/β-catenin signaling was associatedwith decreased cell proliferation (Chien et al., 2009, PNAS,106:1193-1198).

Chemotherapy is a well-established therapeutic approach for numerouscancers, but its efficacy can be limited by side effects and/ortoxicity. In addition, targeted therapies such as the anti-ErbB2receptor (HER2) antibody trastuzumab (HERCEPTIN), tyrosine kinaseinhibitors imatinib (GLEEVEC), dasatinib (SPRYCEL), nilotibib (TASIGNA),sunitinib (SUTENT), sorafenib (NEXAVAR), the anti-VEGF antibodybevacizumab (AVASTIN), and anti-angiogenesis drugs sunitinib (SUTENT)and sorafenib (NEXAVAR), are known to cause, or are likely to cause,side effects and/or toxicity in subjects who take them. Thus, newmethods to identify drug-induced side effects, monitor those sideeffects, and/or mitigate those side effects so that effective cancertherapy can continue are still needed.

BRIEF SUMMARY OF THE INVENTION

The present invention provides improved methods for treating diseasescomprising administering to a subject a therapeutically effective amountof a Wnt pathway inhibitor. For example, in one aspect the inventionprovides methods of screening for, detecting, identifying, monitoring,reducing, preventing, attenuating, and/or mitigating a skeletal-relatedside effect and/or toxicity related to treatment with a Wnt pathwayinhibitor. In some embodiments, the methods comprise determining thelevel of a bone turnover marker in a sample from a patient who hasreceived, is receiving, will receive, or is being considered for initialor further treatment with a Wnt pathway inhibitor, including but notlimited to an anti-Frizzled (FZD) antibody or a soluble FZD receptor.

In another aspect, the invention provides methods of identifying asubject as eligible for treatment with a Wnt pathway inhibitor,comprising: obtaining a biological sample from the subject, determiningthe level of a biomarker in the sample, and identifying the subject aseligible for treatment with the Wnt pathway inhibitor if the level ofthe biomarker is below a predetermined level. In some embodiments, thebiomarker is a bone turnover marker. In some embodiments, the biomarkeris a bone resorption biomarker. In some embodiments, the method ofidentifying a subject as eligible for treatment with a Wnt pathwayinhibitor, comprises: obtaining a biological sample from the subject,determining the level of a bone resorption biomarker in the sample, andidentifying the subject as eligible for treatment with the Wnt pathwayinhibitor if the level of the bone resorption biomarker is below apredetermined level. In some embodiments, the bone resorption biomarkeris collagen type 1 cross-linked C-telopeptide (β-CTX).

In one aspect, the invention provides methods of monitoring a subjectreceiving treatment with a Wnt pathway inhibitor for the development ofskeletal-related side effects and/or toxicity, comprising: obtaining abiological sample from the subject receiving treatment, determining thelevel of a biomarker in the sample, and comparing the level of thebiomarker in the sample to a predetermined level of the biomarker,wherein an increase in the level of the biomarker indicates developmentof skeletal-related side effects and/or toxicity. In some embodiments,the biomarker is a bone turnover marker. In some embodiments, thebiomarker is a bone resorption biomarker. In some embodiments, themethod of monitoring a subject receiving treatment with a Wnt pathwayinhibitor for the development of skeletal-related side effects and/ortoxicity, comprises: obtaining a biological sample from the subjectreceiving treatment, determining the level of a bone resorptionbiomarker in the sample, and comparing the level of the bone resorptionbiomarker in the sample to a predetermined level of the bone resorptionbiomarker, wherein an increase in the level of the bone resorptionbiomarker indicates development of skeletal-related side effects and/ortoxicity. In some embodiments, the bone resorption biomarker is β-CTX.

In another aspect, the invention provides methods of detecting thedevelopment of skeletal-related side effects and/or toxicity in asubject receiving treatment with a Wnt pathway inhibitor, comprising:obtaining a biological sample from the subject receiving treatment,determining the level of a biomarker in the sample, and comparing thelevel of the biomarker in the sample to a predetermined level of thebiomarker, wherein an increase in the level of the biomarker indicatesdevelopment of skeletal-related side effects and/or toxicity. In someembodiments, the biomarker is a bone turnover marker. In someembodiments, the biomarker is a bone resorption biomarker. In someembodiments, the method of detecting the development of askeletal-related side effect and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor, comprises: obtaining abiological sample from the subject receiving treatment, determining thelevel of a bone resorption biomarker in the sample, and comparing thelevel of the bone resorption biomarker in the sample to a predeterminedlevel of the bone resorption biomarker, wherein an increase in the levelof the bone resorption biomarker indicates development of askeletal-related side effect and/or toxicity. In some embodiments, thebone resorption biomarker is β-CTX.

In another aspect, the invention provides methods for identifyingskeletal-related side effects and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor, comprising: obtaining abiological sample from the subject receiving treatment, determining thelevel of a biomarker in the sample, and comparing the level of thebiomarker in the sample to a predetermined level of the biomarker,wherein if the level of the biomarker in the sample is higher than thepredetermined level of the biomarker then a skeletal-related side effectand/or toxicity is indicated. In some embodiments, the biomarker is abone turnover marker. In some embodiments, the biomarker is a boneresorption biomarker. In some embodiments, the method for identifyingskeletal-related side effects and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor, comprises: obtaining abiological sample from the subject receiving treatment, determining thelevel of a bone resorption biomarker in the sample, and comparing thelevel of the bone resorption biomarker in the sample to a predeterminedlevel of the bone resorption biomarker, wherein if the level of the boneresorption biomarker in the sample is higher than the predeterminedlevel of the bone resorption biomarker then a skeletal-related sideeffect and/or toxicity is indicated. In some embodiments, the boneresorption biomarker is β-CTX.

In another aspect, the invention provides methods for monitoringskeletal-related side effects and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor, comprising: obtaining abiological sample from the subject receiving treatment, determining thelevel of a biomarker in the sample, and comparing the level of thebiomarker in the sample to a predetermined level of the biomarker,wherein if the level of the biomarker in the sample is higher than thepredetermined level of the biomarker then a skeletal-related side effectand/or toxicity is indicated. In some embodiments, the biomarker is abone turnover marker. In some embodiments, the biomarker is a boneresorption biomarker. In some embodiments, the method for monitoringskeletal-related side effects and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor, comprises: obtaining abiological sample from the subject receiving treatment, determining thelevel of a bone resorption biomarker in the sample, and comparing thelevel of the bone resorption biomarker in the sample to a predeterminedlevel of the bone resorption biomarker, wherein if the level of the boneresorption biomarker in the sample is higher than the predeterminedlevel of the bone resorption biomarker then a skeletal-related sideeffect and/or toxicity is indicated. In some embodiments, the boneresorption biomarker is β-CTX.

In some aspects and/or embodiments of the methods described herein,wherein if the bone resorption biomarker level (e.g., β-CTX) in a sampleincreases 2-fold or greater as compared to a predetermined level, thesubject is administered a therapeutically effective amount of ananti-resorptive medication. In some embodiments, the bone resorptionbiomarker is β-CTX and the predetermined level is less than about 1000pg/ml. In some embodiments, the anti-resorptive medication is abisphosphonate.

In another aspect, the invention provides methods of reducingskeletal-related side effects and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor, comprising: obtaining abiological sample from the subject receiving treatment, determining thelevel of a bone resorptive biomarker in the sample, comparing the levelof the bone resorptive biomarker in the sample to a predetermined levelof the bone resorptive biomarker, and administering to the subject atherapeutically effective amount of an anti-resorptive medication if thelevel of the bone resorptive biomarker in the sample is higher than thepredetermined level of the bone resorptive biomarker. In someembodiments, the increase in the resorptive biomarker is about 1.5-foldor greater, about 2-fold or greater, about 2.5-fold or greater, or about3-fold or greater than the predetermined level of the bone resorptivebiomarker. In some embodiments, the bone resorption biomarker is β-CTX.In some embodiments, the anti-resorptive medication is a bisphosphonate.

In another aspect, the invention provides methods of preventing orattenuating the development of skeletal-related side effects and/ortoxicity in a subject receiving treatment with a Wnt pathway inhibitor,comprising: obtaining a biological sample from the subject prior totreatment with the Wnt pathway inhibitor, determining the level of abone resorptive biomarker in the sample, comparing the level of the boneresorptive biomarker in the sample to a predetermined level of the boneresorptive biomarker, administering to the subject a therapeuticallyeffective amount of an anti-resorptive medication, and administering tothe subject the Wnt pathway inhibitor. In some embodiments, the boneresorption biomarker is β-CTX. In some embodiments, the anti-resorptivemedication is a bisphosphonate.

In another aspect, the invention provides methods of amelioratingskeletal-related side effects and/or toxicity in a subject administereda Wnt pathway inhibitor, comprising: determining the level of a boneresorptive biomarker in a sample, and administering to the subject atherapeutically effective amount of an anti-resorptive medication. Insome embodiments, the bone resorption biomarker is β-CTX. In someembodiments, the anti-resorptive medication is a bisphosphonate.

In another aspect, the invention provides methods of screening a subjectfor the risk of skeletal-related side effects and/or toxicity fromtreatment with a Wnt pathway inhibitor, comprising: obtaining abiological sample from the subject prior to treatment with the Wntpathway inhibitor, determining the level of a bone resorption biomarkerin the sample, and comparing the level of the bone resorption biomarkerin the sample to a predetermined level of the bone resorption biomarker,wherein if the level of the bone resorption biomarker in the sample ishigher than the predetermined level then the subject is at risk forskeletal-related side effects and/or toxicity. In some embodiments, ifthe subject is at risk for skeletal-related side effects and/ortoxicity, the subject is administered a therapeutically effective amountof a therapeutic agent directed to the skeletal-related side effectand/or toxicity prior to treatment with the Wnt pathway inhibitor. Insome embodiments, the bone resorption biomarker is β-CTX. In someembodiments, the therapeutic agent directed to skeletal-related sideeffects is a bisphosphonate.

In another aspect, the invention provides methods of treating cancer ina subject, comprising: administering to the subject a therapeuticallyeffective amount of a Wnt pathway inhibitor, and determining the levelof a bone resorption biomarker in a sample from the subject. In someembodiments, the method of treating cancer further comprises comparingthe level of the bone resorption biomarker in the sample to apredetermined level of the bone resorption biomarker. In someembodiments, the method of treating cancer further comprises comparingthe level of the bone resorption biomarker in the sample to apredetermined level of the bone resorption biomarker, wherein if thelevel of the bone resorption biomarker is higher than the predeterminedlevel of the bone resorption biomarker then the subject is at risk for askeletal-related side effect and/or toxicity. In some embodiments, themethod of treating cancer further comprises comparing the level of thebone resorption biomarker in the sample to a predetermined level of thebone resorption biomarker, wherein if the level of the bone resorptionbiomarker is higher than the predetermined level of the bone resorptionbiomarker then the subject is administered a therapeutically effectiveamount of an anti-resorptive medication. In some embodiments, the boneresorption biomarker is β-CTX. In some embodiments, the anti-resorptivemedication is a bisphosphonate.

In another aspect, the invention provides methods of inhibiting tumorgrowth in a subject, comprising: administering to the subject atherapeutically effective amount of a Wnt pathway inhibitor, anddetermining the level of a bone resorption biomarker in a sample fromthe subject. In some embodiments, the method of inhibiting tumor growthfurther comprises comparing the level of the bone resorption biomarkerin the sample to a predetermined level of the bone resorption biomarker.In some embodiments, the method of inhibiting tumor growth furthercomprises comparing the level of the bone resorption biomarker in thesample to a predetermined level of the bone resorption biomarker,wherein if the level of the bone resorption biomarker is higher than thepredetermined level of the bone resorption biomarker then the subject isat risk for a skeletal-related side effect and/or toxicity. In someembodiments, the method of inhibiting tumor growth further comprisescomparing the level of the bone resorption biomarker in the sample to apredetermined level of the bone resorption biomarker, wherein if thelevel of the bone resorption biomarker is higher than the predeterminedlevel of the bone resorption biomarker then the subject is administereda therapeutically effective amount of an anti-resorptive medication. Insome embodiments, the bone resorption biomarker is β-CTX. In someembodiments, the anti-resorptive medication is a bisphosphonate.

In some aspects and/or embodiments of the methods described herein, thebiological sample is blood, serum, or plasma. In some embodiments, thebiological sample is a “fasting sample”. As used herein, a “fastingsample” refers to a sample taken from an individual who has not eatenfood and drink anything for at least 9-12 hours. In some embodiments,the predetermined level is about 1500 pg/ml or less in a blood, serum,or plasma sample. In some embodiments, the predetermined level is about1200 pg/ml or less in a blood, serum, or plasma sample. In someembodiments, the predetermined level is about 1000 pg/ml or less in ablood, serum, or plasma sample. In some embodiments, the predeterminedlevel is about 800 pg/ml or less in a blood, serum, or plasma sample. Insome embodiments, the predetermined level is about 600 pg/ml or less ina blood, serum, or plasma sample. In some embodiments, the predeterminedlevel is about 400 pg/ml or less in a blood, serum, or plasma sample. Insome embodiments, the predetermined level of a biomarker (e.g., a boneturnover marker) is the amount of the biomarker in a sample obtained atan earlier date. In some embodiments, the predetermined level of abiomarker (e.g., a bone turnover marker) is the amount of the biomarkerin a sample obtained prior to treatment. In some embodiments, thepredetermined level of a biomarker (e.g., a bone turnover marker) is theamount of the biomarker in a sample obtained at an initial screening. Insome embodiments, the predetermined level of a biomarker (e.g., a boneturnover marker) is a normal reference level. In some embodiments, thepredetermined level of a biomarker is a baseline level. In someembodiments, the baseline level is the amount of the biomarkerdetermined at an initial screening. In some embodiments the boneresorption biomarker is β-CTX. In some embodiments, the predeterminedlevel for β-CTX is about 1000 pg/ml or less in blood, serum, or plasma.

In some aspects and/or embodiments of the methods described herein, abiological sample is obtained approximately every week, every 2 weeks,every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks.

In certain embodiments of each of the aforementioned aspects, as well asother aspects and embodiments described elsewhere herein, the Wntpathway inhibitor is an antibody that specifically binds at least onehuman Wnt protein. Non-limiting examples of anti-Wnt antibodies havebeen described in, for example, U.S. Patent Publication No. 2012/0027778and International Publication WO 2011/088127. In some embodiments, theWnt pathway inhibitor is an antibody that specifically binds at leastone human FZD protein. Non-limiting examples of anti-FZD antibodies havebeen described in, for example, U.S. Pat. No. 7,982,013. In someembodiments, the Wnt pathway inhibitor is a soluble FZD receptor.Non-limiting examples of soluble FZD receptors have been described in,for example, U.S. Pat. Nos. 7,723,477 and 8,324,361 and U.S. PatentPublication No. 2011/0305695.

In some embodiments, the Wnt pathway inhibitor is an antibodycomprising: (a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1),a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and aheavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), and/or (b) a lightchain CDR1 comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2comprising DKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprisingQSYANTLSL (SEQ ID NO:6).

In certain embodiments of each of the aforementioned aspects, as well asother aspects and embodiments described elsewhere herein, the Wntpathway inhibitor is an antibody comprising (a) a heavy chain variableregion having at least about 90%, at least about 95%, or 100% sequenceidentity to SEQ ID NO:7; and/or (b) a light chain variable region havingat least about 90%, at least about 95%, or 100% sequence identity to SEQID NO:8. In some embodiments, the Wnt pathway inhibitor is antibodyOMP-18R5.

In certain embodiments of each of the aforementioned aspects, as well asother aspects and embodiments described elsewhere herein, the Wntpathway inhibitor is a recombinant antibody. In some embodiments, theantibody is a monoclonal antibody, a chimeric antibody, a humanizedantibody, or a human antibody. In some embodiments, the antibody is anantibody fragment comprising an antigen-binding site. In certainembodiments, the antibody or antibody fragment is monovalent,monospecific, or bivalent. In some embodiments, the antibody is abispecific antibody or a multispecific antibody. In some embodiments,the antibody is an IgG1 antibody. In some embodiments, the antibody isan IgG2 antibody. In certain embodiments, the antibody is isolated. Inother embodiments, the antibody is substantially pure.

In some embodiments, the Wnt pathway inhibitor is an antibody that bindsat least one human FZD with a dissociation constant (K_(D)) of about 10nM to about 0.1 nM.

In certain embodiments, the Wnt pathway inhibitor comprises the sameheavy and light chain amino acid sequences as an antibody encoded by aplasmid deposited with ATCC having deposit no. PTA-9541. In certainembodiments, the Wnt pathway inhibitor is encoded by the plasmid havingATCC deposit no. PTA-9541 which was deposited with American Type CultureCollection (ATCC), at 10801 University Boulevard, Manassas, Va., 20110,under the conditions of the Budapest Treaty on Sep. 29, 2008. In certainembodiments, the Wnt pathway inhibitor competes for specific binding toa human FZD with an antibody encoded by the plasmid deposited with ATCChaving deposit no. PTA-9541.

In any of the aspects and/or embodiments of the methods describedherein, the subject has cancer. In some embodiments, the cancer isselected from the group consisting of: lung cancer, pancreatic cancer,breast cancer, colon cancer, colorectal cancer, melanoma,gastrointestinal cancer, gastric cancer, renal cancer, ovarian cancer,liver cancer, endometrial cancer, kidney cancer, prostate cancer,thyroid cancer, neuroblastoma, glioma, glioblastoma multiforme, cervicalcancer, stomach cancer, bladder cancer, hepatoma, and head and neckcancer.

In any of the aspects and/or embodiments of the methods describedherein, the subject is treated with the Wnt pathway inhibitor incombination with one or more additional anti-cancer agents.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but also eachmember of the group individually and all possible subgroups of the maingroup, and also the main group absent one or more of the group members.The present invention also envisages the explicit exclusion of one ormore of any of the group members in the claimed invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Inhibition of breast tumor growth in vivo with intermittentdosing of a Wnt pathway inhibitor. Mice were treated with paclitaxel(-•-), 5 mg/kg OMP-18R5 in combination with paclitaxel (-▪-), 10 mg/kgOMP-18R5 in combination with paclitaxel (-▴-), 25 mg/kg OMP-18R5 incombination with paclitaxel (-▾-), or 45 mg/kg OMP-18R5 in combinationwith paclitaxel (-♦-). Data is shown as tumor volume (mm³) over dayspost-treatment. OMP-18R5 was administered intraperitoneally once everythree weeks (indicated by arrows) and paclitaxel was administered at 10mg/kg once a week.

FIG. 2. Inhibition of breast tumor growth in vivo with intermittentdosing of a Wnt pathway inhibitor. Mice were treated with paclitaxel(-▪-), 25 mg/kg OMP-18R5 in combination with paclitaxel once every 4weeks (-▾-), 25 mg/kg OMP-18R5 in combination with paclitaxel once every2 weeks (-▴-), or 25 mg/kg OMP-18R5 in combination with paclitaxel oncea week (-•-). Data is shown as tumor volume (mm³) over dayspost-treatment. OMP-18R5 was administered intraperitoneally andpaclitaxel was administered at 15 mg/kg once a week.

FIG. 3. Effect of OMP-18R5 on bone formation in mice.

FIG. 4. Effect of zolendronic acid on bone formation in mice treatedwith OMP-18R5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to treating diseases with a Wnt pathwayinhibitor. More particularly, the invention provides methods fortreating cancer comprising administering a Wnt pathway inhibitor, eitheralone or in combination with other anti-cancer agents, and monitoringfor skeletal-related side effects and/or toxicity, including thoserelated to the Wnt pathway inhibitor.

The anti-FZD antibody OMP-18R5 was administered to subjects in a Phase 1single agent dose escalation trial. The data from this early trial, aswell as results from animal studies suggested that administration of aWnt pathway inhibitor such as an anti-FZD antibody may result inskeletal-related side effects and/or toxicity in certain patients.Furthermore, the study showed that increased β-CTX levels may be anearly indicator that a patient being treated with a Wnt pathwayinhibitor is at risk of developing skeletal-related side effects and/ortoxicities, allowing for intervention with appropriate medications.

These results made it desirable to develop risk mitigation andmonitoring strategies for skeletal-related side effects and/ortoxicities as described herein for subjects receiving treatment with aWnt pathway inhibitor (e.g., an anti-FZD antibody or a soluble FZDreceptor) as a single agent or in combination with additionalanti-cancer agents.

I. DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The terms “antagonist” and “antagonistic” as used herein refer to anymolecule that partially or fully blocks, inhibits, reduces, orneutralizes a biological activity of a target and/or signaling pathway(e.g., the Wnt pathway). The term “antagonist” is used herein to includeany molecule that partially or fully blocks, inhibits, reduces, orneutralizes the activity of a protein (e.g., a FZD protein or a Wntprotein). Suitable antagonist molecules specifically include, but arenot limited to, antagonist antibodies, antibody fragments, solublereceptors, or small molecules.

The terms “modulation” and “modulate” as used herein refer to a changeor an alteration in a biological activity. Modulation includes, but isnot limited to, stimulating or inhibiting an activity. Modulation may bean increase or a decrease in activity (e.g., a decrease in Wnt pathwaysignaling), a change in binding characteristics, or any other change inthe biological, functional, or immunological properties associated withthe activity of a protein, pathway, or other biological point ofinterest.

The term “antibody” as used herein refers to an immunoglobulin moleculethat recognizes and specifically binds a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing, through at least one antigen recognitionsite within the variable region of the immunoglobulin molecule. As usedherein, the term encompasses intact polyclonal antibodies, intactmonoclonal antibodies, single chain antibodies, antibody fragments (suchas Fab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv)antibodies, multispecific antibodies such as bispecific antibodies,monospecific antibodies, monovalent antibodies, chimeric antibodies,humanized antibodies, human antibodies, fusion proteins comprising anantigen-binding site of an antibody, and any other modifiedimmunoglobulin molecule comprising an antigen recognition site (e.g.,antigen-binding site) as long as the antibodies exhibit the desiredbiological activity. An antibody can be any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well-known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules, including but not limited to, toxins andradioisotopes.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, singlechain antibodies, and multispecific antibodies formed from antibodyfragments. “Antibody fragment” as used herein comprises anantigen-binding site or epitope-binding site.

The term “variable region” of an antibody refers to the variable regionof an antibody light chain, or the variable region of an antibody heavychain, either alone or in combination. The variable regions of the heavyand light chains each consist of four framework regions (FR) connectedby three complementarity determining regions (CDRs), also known as“hypervariable regions”. The CDRs in each chain are held together inclose proximity by the framework regions and, with the CDRs from theother chain, contribute to the formation of the antigen-binding sites ofthe antibody. There are at least two techniques for determining CDRs:(1) an approach based on cross-species sequence variability (i.e., Kabatet al., 1991, Sequences of Proteins of Immunological Interest, 5thEdition, National Institutes of Health, Bethesda Md.), and (2) anapproach based on crystallographic studies of antigen-antibody complexes(Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948). In addition,combinations of these two approaches are sometimes used in the art todetermine CDRs.

The term “monoclonal antibody” as used herein refers to a homogeneousantibody population involved in the highly specific recognition andbinding of a single antigenic determinant or epitope. This is incontrast to polyclonal antibodies that typically include a mixture ofdifferent antibodies directed against a variety of different antigenicdeterminants. The term “monoclonal antibody” encompasses both intact andfull-length monoclonal antibodies as well as antibody fragments (e.g.,Fab, Fab′, F(ab′)2, Fv), single chain (scFv) antibodies, fusion proteinscomprising an antibody portion, and any other modified immunoglobulinmolecule comprising an antigen recognition site (antigen-binding site).Furthermore, “monoclonal antibody” refers to such antibodies made by anynumber of techniques, including but not limited to, hybridomaproduction, phage selection, recombinant expression, and transgenicanimals.

The term “humanized antibody” as used herein refers to forms ofnon-human (e.g., murine) antibodies that are specific immunoglobulinchains, chimeric immunoglobulins, or fragments thereof that containminimal non-human sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues of the CDRs are replaced by residuesfrom the CDRs of a non-human species (e.g., mouse, rat, rabbit, orhamster) that have the desired specificity, affinity, and/or bindingcapability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al.,1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science,239:1534-1536). In some instances, the Fv framework region residues of ahuman immunoglobulin are replaced with the corresponding residues in anantibody from a non-human species that has the desired specificity,affinity, and/or binding capability. The humanized antibody can befurther modified by the substitution of additional residues either inthe Fv framework region and/or within the replaced non-human residues torefine and optimize antibody specificity, affinity, and/or bindingcapability. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two or three, variabledomains containing all or substantially all of the CDRs that correspondto the non-human immunoglobulin whereas all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody can also comprise at least a portion ofan immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin.

The term “human antibody” as used herein refers to an antibody producedby a human or an antibody having an amino acid sequence corresponding toan antibody produced by a human. A human antibody may be made using anyof the techniques known in the art. This definition of a human antibodyspecifically excludes a humanized antibody comprising non-human CDRs.

The term “chimeric antibody” as used herein refers to an antibodywherein the amino acid sequence of the immunoglobulin molecule isderived from two or more species. Typically, the variable region of bothlight and heavy chains corresponds to the variable region of antibodiesderived from one species of mammals (e.g., mouse, rat, rabbit, etc.)with the desired specificity, affinity, and/or binding capability, whilethe constant regions correspond to sequences in antibodies derived fromanother species (usually human).

The phrase “affinity-matured antibody” as used herein refers to anantibody with one or more alterations in one or more CDRs thereof thatresult in an improvement in the affinity of the antibody for antigen,compared to a parent antibody that does not possess thosealterations(s). The definition also includes alterations in non-CDRresidues made in conjunction with alterations to CDR residues. Preferredaffinity-matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. Affinity-matured antibodies areproduced by procedures known in the art. For example, Marks et al.,1992, Bio/Technology 10:779-783, describes affinity maturation by VH andVL domain shuffling. Random mutagenesis of CDR and/or framework residuesis described by Barbas et al., 1994, PNAS, 91:3809-3813; Schier et al.,1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004;Jackson et al., 1995, J. Immunol., 154:3310-9; and Hawkins et al., 1992,J. Mol. Biol., 226:889-896. Site-directed mutagenesis may also be usedto obtain affinity-matured antibodies.

The terms “epitope” and “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids (also referredto as linear epitopes) are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding (also referred to asconformational epitopes) are typically lost upon protein denaturing. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation.

The terms “selectively binds” or “specifically binds” mean that abinding agent or an antibody reacts or associates more frequently, morerapidly, with greater duration, with greater affinity, or with somecombination of the above to the epitope, protein, or target moleculethan with alternative substances, including unrelated or relatedproteins. In certain embodiments “specifically binds” means, forinstance, that an antibody binds a protein with a K_(D) of about 0.1 mMor less, but more usually less than about 1 μM. In certain embodiments,“specifically binds” means that an antibody binds a target at times witha K_(D) of at least about 0.1 μM or less, at other times at least about0.01 μM or less, and at other times at least about 1 nM or less. Becauseof the sequence identity between homologous proteins in differentspecies, specific binding can include an antibody that recognizes aprotein in more than one species (e.g., human FZD and mouse FZD).Likewise, because of homology within certain regions of polypeptidesequences of different proteins, specific binding can include anantibody (or other polypeptide or binding agent) that recognizes morethan one protein. It is understood that, in certain embodiments, anantibody or binding moiety that specifically binds a first target may ormay not specifically bind a second target. As such, “specific binding”does not necessarily require (although it can include) exclusivebinding, i.e. binding to a single target. Thus, an antibody may, incertain embodiments, specifically bind more than one target. In certainembodiments, multiple targets may be bound by the same antigen-bindingsite on the antibody. For example, an antibody may, in certaininstances, comprise two identical antigen-binding sites, each of whichspecifically binds the same epitope on two or more proteins. In someembodiments, an antibody may be multispecific and comprise at least twoantigen-binding sites with differing specificities. By way ofnon-limiting example, a bispecific antibody may comprise oneantigen-binding site that recognizes an epitope on one protein andfurther comprise a second, different antigen-binding site thatrecognizes a different epitope on a second protein. Generally, but notnecessarily, reference to binding means specific binding.

As used herein the term “soluble receptor” refers to an N-terminalextracellular fragment (or a portion thereof) of a receptor proteinpreceding the first transmembrane domain of the receptor that can besecreted from a cell in soluble form.

As used herein the term “FZD soluble receptor” or “soluble FZD receptor”refers to an N-terminal extracellular fragment of a FZD receptor proteinpreceding the first transmembrane domain of the receptor that can besecreted from a cell in soluble form. FZD soluble receptors comprisingthe entire N-terminal extracellular domain (ECD) as well as smallerfragments are encompassed by the term. Thus, FZD soluble receptorscomprising the Fri domain are also included in this term.

The terms “polypeptide” and “peptide” and “protein” are usedinterchangeably herein and refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids), as well as other modifications known in the art. It isunderstood that, because the polypeptides of this invention may be basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains (e.g., dimers).

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein and refer to polymers of nucleotides of any length, and includeDNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a polymer by DNA or RNApolymerase.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity may be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software that may be used to obtain alignments of aminoacid or nucleotide sequences are well-known in the art. These include,but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG WisconsinPackage, and variations thereof. In some embodiments, two nucleic acidsor polypeptides of the invention are substantially identical, meaningthey have at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using a sequence comparisonalgorithm or by visual inspection. In some embodiments, identity existsover a region of the sequences that is at least about 10, at least about20, at least about 40-60 residues, at least about 60-80 residues inlength or any integral value therebetween. In some embodiments, identityexists over a longer region than 60-80 residues, such as at least about80-100 residues, and in some embodiments the sequences are substantiallyidentical over the full length of the sequences being compared, such asthe coding region of a nucleotide sequence.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Preferably,conservative substitutions in the sequences of the polypeptides andantibodies of the invention do not abrogate the binding of thepolypeptide or antibody containing the amino acid sequence, to theantigen(s), i.e., the one or more RSPO protein(s) to which thepolypeptide or antibody binds. Methods of identifying nucleotide andamino acid conservative substitutions which do not eliminate antigenbinding are well-known in the art.

The term “vector” as used herein means a construct, which is capable ofdelivering, and usually expressing, one or more gene(s) or sequence(s)of interest in a host cell. Examples of vectors include, but are notlimited to, viral vectors, naked DNA or RNA expression vectors, plasmid,cosmid, or phage vectors, DNA or RNA expression vectors associated withcationic condensing agents, and DNA or RNA expression vectorsencapsulated in liposomes.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells, orcompositions include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someembodiments, a polypeptide, antibody, polynucleotide, vector, cell, orcomposition which is isolated is substantially pure.

The term “substantially pure” as used herein refers to material which isat least 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The terms “cancer” and “cancerous” as used herein refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, blastoma, sarcoma, andhematologic cancers such as lymphoma and leukemia.

The terms “tumor” and “neoplasm” as used herein refer to any mass oftissue that results from excessive cell growth or proliferation, eitherbenign (non-cancerous) or malignant (cancerous) including pre-cancerouslesions.

The term “metastasis” as used herein refers to the process by which acancer spreads or transfers from the site of origin to other regions ofthe body with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates (e.g., via thebloodstream or lymph) from the primary site of disease to invadeneighboring body structures.

The terms “cancer stem cell” and “CSC” and “tumor stem cell” and “tumorinitiating cell” are used interchangeably herein and refer to cells froma cancer or tumor that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more types ofdifferentiated cell progeny wherein the differentiated cells havereduced proliferative or developmental potential; and (3) are capable ofsymmetric cell divisions for self-renewal or self-maintenance. Theseproperties confer on the cancer stem cells the ability to form orestablish a tumor or cancer upon serial transplantation into animmunocompromised host (e.g., a mouse) compared to the majority of tumorcells that fail to form tumors. Cancer stem cells undergo self-renewalversus differentiation in a chaotic manner to form tumors with abnormalcell types that can change over time as mutations occur.

The terms “cancer cell” and “tumor cell” refer to the total populationof cells derived from a cancer or tumor or pre-cancerous lesion,including both non-tumorigenic cells, which comprise the bulk of thecancer cell population, and tumorigenic stem cells (cancer stem cells).As used herein, the terms “cancer cell” or “tumor cell” will be modifiedby the term “non-tumorigenic” when referring solely to those cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “tumorigenic” as used herein refers to the functional featuresof a cancer stem cell including the properties of self-renewal (givingrise to additional tumorigenic cancer stem cells) and proliferation togenerate all other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells).

The term “tumorigenicity” as used herein refers to the ability of arandom sample of cells from the tumor to form palpable tumors uponserial transplantation into immunocompromised hosts (e.g., mice). Thisdefinition also includes enriched and/or isolated populations of cancerstem cells that form palpable tumors upon serial transplantation intoimmunocompromised hosts (e.g., mice).

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to, humans, non-human primates, canines, felines, rodents,and the like, which is to be the recipient of a particular treatment.Typically, the terms “subject” and “patient” are used interchangeablyherein in reference to a human subject.

The term “pharmaceutically acceptable” refers to a product or compoundapproved (or approvable) by a regulatory agency of the Federalgovernment or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, includinghumans.

The terms “pharmaceutically acceptable excipient, carrier or adjuvant”or “acceptable pharmaceutical carrier” refer to an excipient, carrier oradjuvant that can be administered to a subject, together with at leastone binding agent (e.g., an antibody) of the present disclosure, andwhich does not destroy the activity of the binding agent. The excipient,carrier, or adjuvant should be non-toxic when administered with abinding agent in doses sufficient to deliver a therapeutic effect.

The terms “effective amount” or “therapeutically effective amount” or“therapeutic effect” refer to an amount of a binding agent, an antibody,polypeptide, polynucleotide, small organic molecule, or other drugeffective to “treat” a disease or disorder in a subject or mammal. Inthe case of cancer, the therapeutically effective amount of a drug(e.g., an antibody) has a therapeutic effect and as such can reduce thenumber of cancer cells; decrease tumorigenicity, tumorigenic frequency,or tumorigenic capacity; reduce the number or frequency of cancer stemcells; reduce the tumor size; reduce the cancer cell population; inhibitand/or stop cancer cell infiltration into peripheral organs including,for example, the spread of cancer into soft tissue and bone; inhibitand/or stop tumor or cancer cell metastasis; inhibit and/or stop tumoror cancer cell growth; relieve to some extent one or more of thesymptoms associated with the cancer; reduce morbidity and mortality;improve quality of life; or a combination of such effects. To the extentthe agent, for example an antibody, prevents growth and/or killsexisting cancer cells, it can be referred to as cytostatic and/orcytotoxic.

The terms “treating” or “treatment” or “to treat” or “alleviating” or“to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent or slow the development of a targeted pathologiccondition or disorder. Thus those in need of treatment include thosealready with the disorder; those prone to have the disorder; and thosein whom the disorder is to be prevented. In some embodiments, a subjectis successfully “treated” according to the methods of the presentinvention if the patient shows one or more of the following: a reductionin the number of or complete absence of cancer cells; a reduction in thetumor size; inhibition of or an absence of cancer cell infiltration intoperipheral organs including the spread of cancer cells into soft tissueand bone; inhibition of or an absence of tumor or cancer cellmetastasis; inhibition or an absence of cancer growth; relief of one ormore symptoms associated with the specific cancer; reduced morbidity andmortality; improvement in quality of life; reduction in tumorigenicity;reduction in the number or frequency of cancer stem cells; or somecombination of effects.

As used in the present disclosure and claims, the singular forms “a”,“an” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising” otherwise analogous embodiments described in termsof “consisting of” and/or “consisting essentially of” are also provided.It is also understood that wherever embodiments are described hereinwith the language “consisting essentially of” otherwise analogousembodiments described in terms of “consisting of” are also provided.

As used herein, reference to “about” or “approximately” a value orparameter includes (and describes) embodiments that are directed to thatvalue or parameter. For example, description referring to “about X”includes description of “X”.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

II. WNT PATHWAY INHIBITORS

The present invention provides Wnt pathway inhibitors for use in methodsof inhibiting tumor growth and/or for use in methods of treating cancer.

In certain embodiments, the Wnt pathway inhibitors are agents that bindone or more human Frizzled proteins (FZD). These agents are referred toherein as “FZD-binding agents”. In some embodiments, the FZD-bindingagents specifically bind one, two, three, four, five, six, seven, eight,nine, or ten FZD proteins. In some embodiments, the FZD-binding agentbinds one or more FZD proteins selected from the group consisting ofFZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. In someembodiments, FZD-binding agent binds one or more FZD proteins comprisingFZD1, FZD2, FZD5, FZD7, and/or FZD8. In certain embodiments, FZD-bindingagent binds FZD7. In certain embodiments, FZD-binding agent binds FZD5and/or FZD8. In certain embodiments, the FZD-binding agent specificallybinds FZD1, FZD2, FZD5, FZD7, and FZD8. Non-limiting examples ofFZD-binding agents can be found in U.S. Pat. No. 7,982,013.

In certain embodiments, the FZD-binding agent is a FZD antagonist. Incertain embodiments, the FZD-binding agent is a Wnt pathway antagonist.In certain embodiments, the FZD-binding agent inhibits Wnt signaling. Insome embodiments, the FZD-binding agent inhibits canonical Wntsignaling.

In some embodiments, the FZD-binding agents are antibodies. In someembodiments, the FZD-binding agents are polypeptides. In certainembodiments, the FZD-binding agent is an antibody or a polypeptidecomprising an antigen-binding site. In certain embodiments, anantigen-binding site of a FZD-binding antibody or polypeptide describedherein is capable of binding (or binds) one, two, three, four, five, ormore human FZD proteins. In certain embodiments, an antigen-binding siteof the FZD-binding antibody or polypeptide is capable of specificallybinding one, two, three, four, or five human FZD proteins selected fromthe group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8,FZD9 and FZD10. In some embodiments, when the FZD-binding agent is anantibody that binds more than one FZD protein, it may be referred to asa “pan-FZD antibody”.

In certain embodiments, the FZD-binding agent (e.g., antibody)specifically binds the extracellular domain (ECD) within the one or morehuman FZD proteins to which it binds. In certain embodiments, theFZD-binding agent specifically binds within the Fri domain (also knownas the cysteine-rich domain (CRD)) of the human FZD protein to which itbinds. Sequences of the Fri domain of each of the human FZD proteins areknown in the art and are provided as SEQ ID NO:13 (FZD1), SEQ ID NO:14(FZD2), SEQ ID NO:15 (FZD3), SEQ ID NO:16 (FZD4), SEQ ID NO:17 (FZD5),SEQ ID NO:18 (FZD6), SEQ ID NO:19 (FZD7), SEQ ID NO:20 (FZD), SEQ IDNO:21 (FZD9), and SEQ ID NO:22 (FZD10).

In certain embodiments, the FZD-binding agent binds one, two, three,four, five, or more FZD proteins. In some embodiments, the FZD-bindingagent specifically binds one, two, three, four, or five FZD proteinsselected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8.In some embodiments, the FZD-binding agent specifically binds at leastFZD5 and FZD8.

In some embodiments, the FZD-binding agent binds at least one human FZDprotein with a dissociation constant (K_(D)) of about 1 μM or less,about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10nM or less, about 1 nM or less, or about 0.1 nM or less. In someembodiments, a FZD-binding agent binds at least one FZD protein with aK_(D) of about 10 nM or less. In some embodiments, a FZD-binding agentbinds at least one FZD protein with a K_(D) of about 1 nM or less. Insome embodiments, a FZD-binding agent binds at least one FZD proteinwith a K_(D) of about 0.1 nM or less. In certain embodiments, aFZD-binding agent binds each of one or more (e.g., 1, 2, 3, 4, or 5) ofFZD1, FZD2, FZD5, FZD7, and FZD8 with a K_(D) of about 40 nM or less. Incertain embodiments, the FZD-binding agent binds to each of one or moreof FZD 1, FZD2, FZD5, FZD7, and FZD8 with a K_(D) of about 10 nM orless. In certain embodiments, the FZD-binding agent binds each of FZD 1,FZD2, FZD5, FZD7, and FZD8 with a K_(D) of about 10 nM. In someembodiments, the K_(D) of the binding agent (e.g., an antibody) to a FZDprotein is the K_(D) determined using a FZD-Fc fusion protein comprisingat least a portion of the FZD extracellular domain or FZD-Fri domainimmobilized on a Biacore chip.

In certain embodiments, the FZD-binding agent binds one or more (forexample, two or more, three or more, or four or more) human FZD proteinswith an EC₅₀ of about 1 μM or less, about 100 nM or less, about 40 nM orless, about 20 nM or less, about 10 nM or less, or about 1 nM or less.In certain embodiments, a FZD-binding agent binds to more than one FZDprotein with an EC₅₀ of about 40 nM or less, about 20 nM or less, orabout 10 nM or less. In certain embodiments, the FZD-binding agent hasan EC₅₀ of about 20 nM or less with respect to one or more (e.g., 1, 2,3, 4, or 5) of the following FZD proteins: FZD1, FZD2, FZD5, FZD7, andFZD8. In certain embodiments, the FZD-binding agent has an EC₅₀ of about10 nM or less with respect to one or more (e.g., 1, 2, 3, 4, or 5) ofthe following FZD proteins: FZD1, FZD2, FZD5, FZD7, and FZD8. In certainembodiments, the FZD-binding agent has an EC₅₀ of about 40 nM or less or20 nM or less with respect to binding of FZD5 and/or FZD8.

In certain embodiments, the Wnt pathway inhibitor is a FZD-binding agentwhich is an antibody. In some embodiments, the antibody is a recombinantantibody. In some embodiments, the antibody is a monoclonal antibody. Insome embodiments, the antibody is a chimeric antibody. In someembodiments, the antibody is a humanized antibody. In some embodiments,the antibody is a human antibody. In certain embodiments, the antibodyis an IgG1 antibody. In certain embodiments, the antibody is an IgG2antibody. In certain embodiments, the antibody is an antibody fragmentcomprising an antigen-binding site. In some embodiments, the antibody ismonovalent, monospecific, or bivalent. In some embodiments, the antibodyis a bispecific antibody or a multispecific antibody. In someembodiments, the antibody is conjugated to a cytotoxic moiety. In someembodiments, the antibody is isolated. In some embodiments, the antibodyis substantially pure.

The FZD-binding agents (e.g., antibodies) of the present invention canbe assayed for specific binding by any method known in the art. Theimmunoassays which can be used include, but are not limited to,competitive and non-competitive assay systems using techniques such asBiacore analysis, FACS analysis, immunofluorescence,immunocytochemistry, Western blot analysis, radioimmunoassays, ELISA,“sandwich” immunoassays, immunoprecipitation assays, precipitationreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, and protein A immunoassays. Suchassays are routine and well-known in the art (see, e.g., Ausubel et al.,Editors, 1994-present, Current Protocols in Molecular Biology, JohnWiley & Sons, Inc., New York, N.Y.).

For example, the specific binding of an antibody to a human FZD proteinmay be determined using ELISA. An ELISA assay comprises preparingantigen, coating wells of a 96 well microtiter plate with antigen,adding to the well the FZD-binding agent (e.g., an antibody) conjugatedto a detectable compound such as an enzymatic substrate (e.g.horseradish peroxidase or alkaline phosphatase), incubating for a periodof time and detecting the presence of the FZD-binding agent bound to theantigen. In some embodiments, the FZD-binding antibody or agent is notconjugated to a detectable compound, but instead a second conjugatedantibody that recognizes the FZD-binding antibody or agent (e.g., ananti-Fc antibody) is added to the well. In some embodiments, instead ofcoating the well with the antigen, the FZD-binding antibody or agent canbe coated to the well and a second antibody conjugated to a detectablecompound can be added following the addition of the antigen to thecoated well. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase and/or optimize the signaldetected as well as other variations of ELISAs that may be used.

In another example, the specific binding of an antibody to a human FZDprotein may be determined using FACS. A FACS screening assay maycomprise generating a cDNA construct that expresses an antigen as afusion protein, transfecting the construct into cells, expressing theantigen on the surface of the cells, mixing the FZD-binding antibody orother FZD-binding agent with the transfected cells, and incubating for aperiod of time. The cells bound by a FZD-binding antibody or otherFZD-binding agent may be identified by using a secondary antibodyconjugated to a detectable compound (e.g., PE-conjugated anti-Fcantibody) and a flow cytometer. One of skill in the art would beknowledgeable as to the parameters that can be modified to optimize thesignal detected as well as other variations of FACS that may enhancescreening (e.g., screening for blocking antibodies).

The binding affinity of an antibody or other binding-agent to an antigen(e.g., a FZD protein) and the off-rate of an antibody-antigeninteraction can be determined by competitive binding assays. One exampleof a competitive binding assay is a radioimmunoassay comprising theincubation of labeled antigen (e.g., ³H or ¹²⁵I), or fragment or variantthereof, with the antibody of interest in the presence of increasingamounts of unlabeled antigen followed by the detection of the antibodybound to the labeled antigen. The affinity of the antibody for anantigen (e.g., a FZD protein) and the binding off-rates can bedetermined from the data by Scatchard plot analysis. In someembodiments, Biacore kinetic analysis is used to determine the bindingon and off rates of antibodies or agents that bind an antigen (e.g., aFZD protein). Biacore kinetic analysis comprises analyzing the bindingand dissociation of antibodies from chips with immobilized antigen(e.g., a FZD protein) on their surface.

In certain embodiments, the invention provides a Wnt pathway inhibitorwhich is a FZD-binding agent (e.g., an antibody) that comprises a heavychain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3comprising NFIKYVFAN (SEQ ID NO:3). In some embodiments, the FZD-bindingagent further comprises a light chain CDR1 comprising SGDNIGSFYVH (SEQID NO:4), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5), and alight chain CDR3 comprising QSYANTLSL (SEQ ID NO:6). In someembodiments, the FZD-binding agent comprises a light chain CDR1comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprisingDKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQID NO:6). In certain embodiments, the FZD-binding agent comprises: (a) aheavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3comprising NFIKYVFAN (SEQ ID NO:3), and (b) a light chain CDR1comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprisingDKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQID NO:6).

In certain embodiments, the invention provides a FZD-binding agent(e.g., an antibody) that comprises: (a) a heavy chain CDR1 comprisingGFTFSHYTLS (SEQ ID NO:1), or a variant thereof comprising 1, 2, 3, or 4amino acid substitutions; (b) a heavy chain CDR2 comprisingVISGDGSYTYYADSVKG (SEQ ID NO:2), or a variant thereof comprising 1, 2,3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprisingNFIKYVFAN (SEQ ID NO:3), or a variant thereof comprising 1, 2, 3, or 4amino acid substitutions; (d) a light chain CDR1 comprising SGDNIGSFYVH(SEQ ID NO:4), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (e) a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5),or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;and (f) a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:6), or avariant thereof comprising 1, 2, 3, or 4 amino acid substitutions. Incertain embodiments, the amino acid substitutions are conservativesubstitutions.

In certain embodiments, the invention provides a FZD-binding agent(e.g., an antibody) that comprises a heavy chain variable region havingat least about 80% sequence identity to SEQ ID NO:7, and/or a lightchain variable region having at least 80% sequence identity to SEQ IDNO:8. In certain embodiments, the FZD-binding agent comprises a heavychain variable region having at least about 85%, at least about 90%, atleast about 95%, at least about 97%, or at least about 99% sequenceidentity to SEQ ID NO:7. In certain embodiments, the FZD-binding agentcomprises a light chain variable region having at least about 85%, atleast about 90%, at least about 95%, at least about 97%, or at leastabout 99% sequence identity to SEQ ID NO:8. In certain embodiments, theFZD-binding agent comprises a heavy chain variable region having atleast about 95% sequence identity to SEQ ID NO:7, and/or a light chainvariable region having at least about 95% sequence identity to SEQ IDNO:8. In certain embodiments, the FZD-binding agent comprises a heavychain variable region comprising SEQ ID NO:7 and/or a light chainvariable region comprising SEQ ID NO:8. In certain embodiments, theFZD-binding agent comprises a heavy chain variable region comprising SEQID NO:7 and a light chain variable region comprising SEQ ID NO:8. Incertain embodiments, the FZD-binding agent comprises a heavy chainvariable region consisting essentially of SEQ ID NO:7 and a light chainvariable region consisting essentially of SEQ ID NO:8.

In certain embodiments, the invention provides a FZD-binding agent(e.g., an antibody) that comprises: (a) a heavy chain having at least90% sequence identity to SEQ ID NO:9 (with or without the signalsequence) or SEQ ID NO:11; and/or (b) a light chain having at least 90%sequence identity to SEQ ID NO:10 (with or without the signal sequence)or SEQ ID NO:12. In some embodiments, the FZD-binding agent comprises:(a) a heavy chain having at least 95% sequence identity to SEQ ID NO:9(with or without the signal sequence) or SEQ ID NO:11; and/or (b) alight chain having at least 95% sequence identity to SEQ ID NO:10 (withor without the signal sequence) or SEQ ID NO:12. In some embodiments,the FZD-binding agent comprises a heavy chain comprising SEQ ID NO:9(with or without the signal sequence) or SEQ ID NO:11, and/or a lightchain comprising SEQ ID NO:10 (with or without the signal sequence) orSEQ ID NO:12. In some embodiments, the FZD-binding agent comprises aheavy chain comprising SEQ ID NO:11 and a light chain comprising SEQ IDNO:12. In some embodiments, the FZD-binding agent comprises a heavychain consisting essentially of amino acids 20-463 of SEQ ID NO:9 and alight chain consisting essentially of amino acids 20-232 of SEQ IDNO:10. In some embodiments, the FZD-binding agent comprises a heavychain consisting essentially of SEQ ID NO:11 and a light chainconsisting essentially of SEQ ID NO:12.

In certain embodiments, the invention provides a Wnt pathway inhibitorwhich is a FZD-binding agent (e.g., an antibody) that specifically bindsat least one of FZD1, FZD2, FZD5, FZD7 and/or FZD8, wherein theFZD-binding agent (e.g., an antibody) comprises one, two, three, four,five, and/or six of the CDRs of antibody 18R5. Antibody 18R5 (also knownas OMP-18R5 and vantictumab), as well as other FZD-binding agents, hasbeen previously described in U.S. Pat. No. 7,982,013. DNA encoding theheavy chain and light chain of the 18R51gG2 antibody was deposited withthe ATCC, under the conditions of the Budapest Treaty on Sep. 29, 2008,and assigned ATCC deposit designation number PTA-9541. In someembodiments, the FZD-binding agent comprises one or more of the CDRs of18R5, two or more of the CDRs of 18R5, three or more of the CDRs of18R5, four or more of the CDRs of 18R5, five or more of the CDRs of18R5, or all six of the CDRs of 18R5.

The invention provides polypeptides which are Wnt pathway inhibitors.The polypeptides include, but are not limited to, antibodies thatspecifically bind human FZD proteins. In some embodiments, a polypeptidebinds one or more FZD proteins selected from the group consisting ofFZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. In someembodiments, a polypeptide binds FZD1, FZD2, FZD5, FZD7, and/or FZD8. Insome embodiments, a polypeptide binds FZD1, FZD2, FZD5, FZD7, and FZD8.

In certain embodiments, a polypeptide comprises one, two, three, four,five, and/or six of the CDRs of antibody 18R5. In some embodiments, apolypeptide comprises CDRs with up to four (i.e., 0, 1, 2, 3, or 4)amino acid substitutions per CDR. In certain embodiments, the heavychain CDR(s) are contained within a heavy chain variable region. Incertain embodiments, the light chain CDR(s) are contained within a lightchain variable region.

In some embodiments, the invention provides a polypeptide thatspecifically binds one or more human FZD proteins, wherein thepolypeptide comprises an amino acid sequence having at least about 80%sequence identity to SEQ ID NO:7, and/or an amino acid sequence havingat least about 80% sequence identity to SEQ ID NO:8. In certainembodiments, the polypeptide comprises an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about97%, or at least about 99% sequence identity to SEQ ID NO:7. In certainembodiments, the polypeptide comprises an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about97%, or at least about 99% sequence identity to SEQ ID NO:8. In certainembodiments, the polypeptide comprises an amino acid sequence having atleast about 95% sequence identity to SEQ ID NO:7, and/or an amino acidsequence having at least about 95% sequence identity to SEQ ID NO:8. Incertain embodiments, the polypeptide comprises an amino acid sequencecomprising SEQ ID NO:7, and/or an amino acid sequence comprising SEQ IDNO:8.

In some embodiments, a FZD-binding agent comprises a polypeptidecomprising a sequence selected from the group consisting of: SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ IDNO:12.

In certain embodiments, a FZD-binding agent comprises the heavy chainvariable region and light chain variable region of the 18R5 antibody. Incertain embodiments, a FZD-binding agent comprises the heavy chain andlight chain of the 18R5 antibody (with or without the leader sequence).

In certain embodiments, a FZD-binding agent comprises, consistsessentially of, or consists of, the antibody 18R5.

In certain embodiments, a FZD-binding agent (e.g., antibody) competesfor specific binding to one or more human FZD proteins with an antibodythat comprises a heavy chain variable region comprising SEQ ID NO:7 anda light chain variable region comprising SEQ ID NO:8. In certainembodiments, a FZD-binding agent (e.g., antibody) competes for specificbinding to one or more human FZD proteins with an antibody thatcomprises a heavy chain comprising SEQ ID NO:9 (with or without thesignal sequence) and a light chain comprising SEQ ID NO:10 (with orwithout the signal sequence). In certain embodiments, a FZD-bindingagent (e.g., antibody) competes for specific binding to one or morehuman FZD proteins with an antibody that comprises a heavy chaincomprising SEQ ID NO:11 and a light chain comprising SEQ ID NO:12. Incertain embodiments, a FZD-binding agent competes with antibody 18R5 forspecific binding to one or more human FZD proteins. In some embodiments,a FZD-binding agent or antibody competes for specific binding to one ormore human FZD proteins in an in vitro competitive binding assay.

In certain embodiments, a FZD-binding agent (e.g., an antibody) bindsthe same epitope, or essentially the same epitope, on one or more humanFZD proteins as an antibody of the invention. In another embodiment, aFZD-binding agent is an antibody that binds an epitope on one or morehuman FZD proteins that overlaps with the epitope on a FZD protein boundby an antibody of the invention. In certain embodiments, a FZD-bindingagent (e.g., an antibody) binds the same epitope, or essentially thesame epitope, on one or more FZD proteins as antibody 18R5. In anotherembodiment, the FZD-binding agent is an antibody that binds an epitopeon one or more human FZD proteins that overlaps with the epitope on aFZD protein bound by antibody 18R5.

In certain embodiments, the Wnt pathway inhibitors are agents that bindone or more human Wnt proteins. These agents are referred to herein as“Wnt-binding agents”. In certain embodiments, the agents specificallybind one, two, three, four, five, six, seven, eight, nine, ten, or moreWnt proteins. In some embodiments, the Wnt-binding agents bind one ormore human Wnt proteins selected from the group consisting of Wnt1,Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a,Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. In certainembodiments, a Wnt-binding agent binds one or more (or two or more,three or more, four or more, five or more, etc.) Wnt proteins selectedfrom the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a,Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. In certain embodiments, the oneor more (or two or more, three or more, four or more, five or more,etc.) Wnt proteins are selected from the group consisting of Wnt1, Wnt2,Wnt2b, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, and Wnt10b.

In certain embodiments, the Wnt-binding agent is a Wnt antagonist. Incertain embodiments, the Wnt-binding agent is a Wnt pathway antagonist.In certain embodiments, the Wnt-binding agent inhibits Wnt signaling. Insome embodiments, the Wnt-binding agent inhibits canonical Wntsignaling.

In some embodiments, the Wnt-binding agent is an antibody. In someembodiments, the Wnt-binding agent is a polypeptide. In certainembodiments, the Wnt-binding agent is an antibody or a polypeptidecomprising an antigen-binding site. In certain embodiments, anantigen-binding site of a Wnt-binding antibody or polypeptide describedherein is capable of binding (or binds) one, two, three, four, five, ormore human Wnt proteins. In certain embodiments, an antigen-binding siteof the Wnt-binding antibody or polypeptide is capable of specificallybinding one, two, three, four, or five human Wnt proteins selected fromthe group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b,Wnt8a, Wnt8b, Wnt10a, and Wnt10b. Non-limiting examples of Wnt-bindingagents can be found in International Publication WO 2011/088127.

In certain embodiments, a Wnt-binding agent binds to the C-terminalcysteine rich domain of one or more human Wnt proteins. In certainembodiments, the Wnt-binding agent binds a domain within the one or moreWnt proteins to which the agent or antibody binds that is selected fromthe group consisting of: SEQ ID NO:46 (Wnt1), SEQ ID NO:47 (Wnt2), SEQID NO:48 (Wnt2b), SEQ ID NO:49 (Wnt3), SEQ ID NO:50 (Wnt3a), SEQ IDNO:51 (Wnt7a), SEQ ID NO:52 (Wnt7b), SEQ ID NO:53 (Wnt8a), SEQ ID NO:54(Wnt8b), SEQ ID NO:55 (Wnt10a), and SEQ ID NO:56 (Wnt10b).

In certain embodiments, the Wnt-binding agent binds one or more (e.g.,two or more, three or more, or four or more) Wnt proteins with a K_(D)of about 1 μM or less, about 100 nM or less, about 40 nM or less, about20 nM or less, or about 10 nM or less. For example, in certainembodiments, a Wnt-binding agent described herein that binds more thanone Wnt protein, binds those Wnt proteins with a K_(D) of about 100 nMor less, about 20 nM or less, or about 10 nM or less. In certainembodiments, the Wnt-binding agent binds each of one or more (e.g., 1,2, 3, 4, or 5) Wnt proteins with a K_(D) of about 40 nM or less, whereinthe Wnt proteins are selected from the group consisting of: Wnt1, Wnt2,Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. Insome embodiments, the K_(D) of the binding agent (e.g., an antibody) toa Wnt protein is the K_(D) determined using a Wnt fusion proteincomprising at least a portion of the Wnt C-terminal cysteine rich domainimmobilized on a Biacore chip.

In certain embodiments, the Wnt-binding agent binds one or more (forexample, two or more, three or more, or four or more) human Wnt proteinswith an EC₅₀ of about 1 μM or less, about 100 nM or less, about 40 nM orless, about 20 nM or less, about 10 nM or less, or about 1 nM or less.In certain embodiments, a Wnt-binding agent binds to more than one Wntwith an EC₅₀ of about 40 nM or less, about 20 nM or less, or about 10 nMor less. In certain embodiments, the Wnt-binding agent has an EC₅₀ ofabout 20 nM or less with respect to one or more (e.g., 1, 2, 3, 4, or 5)of Wnt proteins Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b,Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11,and/or Wnt16. In certain embodiments, the Wnt-binding agent has an EC₅₀of about 10 nM or less with respect to one or more (e.g., 1, 2, 3, 4, or5) of the following Wnt proteins Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8a,Wnt8b, Wnt10a, and/or Wnt10b.

In certain embodiments, the Wnt pathway inhibitor is a Wnt-binding agentwhich is an antibody. In some embodiments, the antibody is a recombinantantibody. In some embodiments, the antibody is a monoclonal antibody. Insome embodiments, the antibody is a chimeric antibody. In someembodiments, the antibody is a humanized antibody. In some embodiments,the antibody is a human antibody. In certain embodiments, the antibodyis an IgG1 antibody. In certain embodiments, the antibody is an IgG2antibody. In certain embodiments, the antibody is an antibody fragmentcomprising an antigen-binding site. In some embodiments, the antibody ismonovalent, monospecific, or bivalent. In some embodiments, the antibodyis a bispecific antibody or a multispecific antibody. In someembodiments, the antibody is conjugated to a cytotoxic moiety. In someembodiments, the antibody is isolated. In some embodiments, the antibodyis substantially pure.

The Wnt-binding agents (e.g., antibodies) of the present invention canbe assayed for specific binding by any method known in the art asdescribed herein for FZD-binding agents.

For example, the specific binding of an antibody to a human Wnt proteinmay be determined using ELISA. An ELISA assay comprises preparingantigen, coating wells of a 96 well microtiter plate with antigen,adding to the well the Wnt-binding agent (e.g., an antibody) conjugatedto a detectable compound such as an enzymatic substrate (e.g.horseradish peroxidase or alkaline phosphatase), incubating for a periodof time and detecting the presence of the Wnt-binding agent bound to theantigen. In some embodiments, the Wnt-binding antibody or agent is notconjugated to a detectable compound, but instead a second conjugatedantibody that recognizes the Wnt-binding antibody or agent (e.g., ananti-Fc antibody) is added to the well. In some embodiments, instead ofcoating the well with the antigen, the Wnt-binding antibody or agent canbe coated to the well and a second antibody conjugated to a detectablecompound can be added following the addition of the antigen to thecoated well. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase and/or optimize the signaldetected as well as other variations of ELISAs that may be used.

In another example, the specific binding of an antibody to a human Wntprotein may be determined using FACS. A FACS screening assay maycomprise generating a cDNA construct that expresses an antigen as afusion protein, transfecting the construct into cells, expressing theantigen on the surface of the cells, mixing the Wnt-binding antibodywith the transfected cells, and incubating for a period of time. Thecells bound by the Wnt-binding antibody may be identified by using asecondary antibody conjugated to a detectable compound (e.g.,PE-conjugated anti-Fc antibody) and a flow cytometer. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto optimize the signal detected as well as other variations of FACS thatmay enhance screening (e.g., screening for blocking antibodies).

The binding affinity of a Wnt-binding agent to an antigen (e.g., a Wntprotein) and the off-rate of an antibody-antigen interaction can bedetermined by competitive binding assays such as those described abovefor FZD-binding agents.

In certain embodiments, the Wnt-binding agent is a soluble receptor. Incertain embodiments, the Wnt-binding agent comprises the extracellulardomain of a FZD receptor protein. In some embodiments, the Wnt-bindingagent comprises a Fri domain of a FZD protein. In some embodiments, asoluble receptor comprising a FZD Fri domain can demonstrate alteredbiological activity (e.g., increased protein half-life) compared to asoluble receptor comprising the entire FZD ECD. Protein half-life can befurther increased by covalent modification with polyethylene glycol(PEG) or polyethylene oxide (PEO). In certain embodiments, the FZDprotein is a human FZD protein. In certain embodiments, the human FZDprotein is FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, orFZD10. Non-limiting examples of soluble FZD receptors can be found inU.S. Pat. Nos. 7,723,477 and 7,947,277; and U.S. Patent Publication No.2011/0305695.

The predicted Fri domains for each of the human FZD1-10 proteins areprovided as SEQ ID NOs:13-22. The predicted minimal Fri domains for eachof the human FZD 1-10 proteins are provided as SEQ ID NOs:23-32. Thoseof skill in the art may differ in their understanding of the exact aminoacids corresponding to the various Fri domains. Thus, the N-terminusand/or C-terminus of the domains outlined above and herein may extend orbe shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids.

In certain embodiments, the Wnt-binding agent comprises a Fri domain ofa human FZD protein, or a fragment or variant of the Fri domain thatbinds one or more human Wnt proteins. In certain embodiments, the humanFZD protein is FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, orFZD10. In certain embodiments, the human FZD protein is FZD4. In certainembodiments, the human FZD protein is FZD5. In certain embodiments, thehuman FZD protein is FZD8. In certain embodiments, the human FZD proteinis FZD10. In certain embodiments, the FZD protein is FZD4 and theWnt-binding agent comprises SEQ ID NO:16. In certain embodiments, theFZD protein is FZD5 and the Wnt-binding agent comprises SEQ ID NO:17. Incertain embodiments, the FZD protein is FZD7 and the Wnt-binding agentcomprises SEQ ID NO:19. In certain embodiments, the FZD protein is FZD8and the Wnt-binding agent comprises SEQ ID NO:20. In certainembodiments, the FZD protein is FZD 10 and the Wnt-binding agentcomprises SEQ ID NO:22. In certain embodiments, the FZD protein is FZD8and the Wnt-binding agent comprises SEQ ID NO:33.

In some embodiments, the Wnt-binding agent comprises a Fri domaincomprising the minimal Fri domain of FZD1 (SEQ ID NO:23), the minimalFri domain of FZD2 (SEQ ID NO:24), the minimal Fri domain of FZD3 (SEQID NO:25), the minimal Fri domain of FZD4 (SEQ ID NO:26), the minimalFri domain of FZD5 (SEQ ID NO:27), the minimal Fri domain of FZD6 (SEQID NO:28), the minimal Fri domain of FZD7 (SEQ ID NO:29), the minimalFri domain of FZD8 (SEQ ID NO:30), the minimal Fri domain of FZD9 (SEQID NO:31), or the minimal Fri domain of FZD10 (SEQ ID NO:32). In someembodiments, the Wnt-binding agent comprises a Fri domain comprising theminimal Fri domain of FZD8 (SEQ ID NO:30).

In some embodiments, the Wnt-binding agent comprises a Fri domainconsisting essentially of the Fri domain of FZD 1, the Fri domain ofFZD2, the Fri domain of FZD3, the Fri domain of FZD4, the Fri domain ofFZD5, the Fri domain of FZD6, the Fri domain of FZD7, the Fri domain ofFZD8, the Fri domain of FZD9, or the Fri domain of FZD10. In someembodiments, the Wnt-binding agent comprises a Fri domain consistingessentially of the Fri domain of FZD8.

In some embodiments, the Wnt-binding agent comprises a sequence selectedfrom the group consisting of: SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. In some embodiments, theWnt-binding agent comprises a Fri domain consisting essentially of SEQID NO:20. In some embodiments, the Wnt-binding agent comprises a Fridomain consisting essentially of SEQ ID NO:33.

In certain embodiments, the Wnt-binding agent comprises a variant of anyone of the aforementioned FZD Fri domain sequences that comprises one ormore (e.g., one, two, three, four, five, six, seven, eight, nine, ten,etc.) conservative substitutions and is capable of binding Wntprotein(s).

In certain embodiments, a Wnt-binding agent, such as an agent comprisinga Fri domain of a human FZD receptor, further comprises a non-FZDpolypeptide. In some embodiments, a FZD soluble receptor may include FZDECD or Fri domains linked to other non-FZD functional and structuralpolypeptides including, but not limited to, a human Fc region, proteintags (e.g., myc, FLAG, GST), other endogenous proteins or proteinfragments, or any other useful protein sequence including any linkerregion between a FZD ECD or Fri domain and a second polypeptide. Incertain embodiments, the non-FZD polypeptide comprises a human Fcregion. The Fc region can be obtained from any of the classes ofimmunoglobulin, IgG, IgA, IgM, IgD and IgE. In some embodiments, the Fcregion is a human IgG1 Fc region. In some embodiments, the Fc region isa human IgG2 Fc region. In some embodiments, the Fc region is awild-type Fc region. In some embodiments, the Fc region is a mutated Fcregion. In some embodiments, the Fc region is truncated at theN-terminal end by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, (e.g.,in the hinge domain). In some embodiments, an amino acid in the hingedomain is changed to hinder undesirable disulfide bond formation. Insome embodiments, a cysteine is replaced with a serine to hinder orblock undesirable disulfide bond formation. In some embodiments, the Fcregion is truncated at the C-terminal end by 1, 2, 3, or more aminoacids. In some embodiments, the Fc region is truncated at the C-terminalend by 1 amino acid. In certain embodiments, the non-FZD polypeptidecomprises SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQID NO:38. In certain embodiments, the non-FZD polypeptide consistsessentially of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,or SEQ ID NO:38. In certain embodiments, the non-FZD polypeptideconsists essentially of SEQ ID NO:36 or SEQ ID NO:37.

In certain embodiments, a Wnt-binding agent is a fusion proteincomprising at least a minimal Fri domain of a FZD receptor and a Fcregion. As used herein, a “fusion protein” is a hybrid protein expressedby a nucleic acid molecule comprising nucleotide sequences of at leasttwo genes. In some embodiments, the C-terminus of the first polypeptideis linked to the N-terminus of the immunoglobulin Fc region. In someembodiments, the first polypeptide (e.g., a FZD Fri domain) is directlylinked to the Fc region (i.e. without an intervening linker). In someembodiments, the first polypeptide is linked to the Fc region via alinker.

As used herein, the term “linker” refers to a linker inserted between afirst polypeptide (e.g., a FZD component) and a second polypeptide(e.g., a Fc region). In some embodiments, the linker is a peptidelinker. Linkers should not adversely affect the expression, secretion,or bioactivity of the polypeptide. Linkers should not be antigenic andshould not elicit an immune response. Suitable linkers are known tothose of skill in the art and often include mixtures of glycine andserine residues and often include amino acids that are stericallyunhindered. Other amino acids that can be incorporated into usefullinkers include threonine and alanine residues. Linkers can range inlength, for example from 1-50 amino acids in length, 1-22 amino acids inlength, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3amino acids in length. Linkers may include, but are not limited to,SerGly, GGSG, GSGS, GGGS, S(GGS)n where n is 1-7, GRA, poly(Gly),poly(Ala), ESGGGGVT (SEQ ID NO:57), LESGGGGVT (SEQ ID NO:58), GRAQVT(SEQ ID NO:59), WRAQVT (SEQ ID NO:60), and ARGRAQVT (SEQ ID NO:61). Asused herein, a linker is an intervening peptide sequence that does notinclude amino acid residues from either the C-terminus of the firstpolypeptide (e.g., a FZD Fri domain) or the N-terminus of the secondpolypeptide (e.g., the Fc region).

In some embodiments, the Wnt-binding agent comprises a FZD Fri domain, aFc region and a linker connecting the FZD Fri domain to the Fc region.In some embodiments, the FZD Fri domain comprises SEQ ID NO:20, SEQ IDNO:30, or SEQ ID NO:33. In some embodiments, the linker comprisesESGGGGVT (SEQ ID NO:57) or LESGGGGVT (SEQ ID NO:58).

In some embodiments, the Wnt-binding agent comprises a first polypeptidecomprising SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, or SEQ ID NO:33; and a second polypeptide comprising SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38,wherein the first polypeptide is directly linked to the secondpolypeptide. In some embodiments, the Wnt-binding agent comprises afirst polypeptide comprising SEQ ID NO:20 and a second polypeptidecomprising SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, orSEQ ID NO:38. In some embodiments, the Wnt-binding agent comprises afirst polypeptide comprising SEQ ID NO:20 and a second polypeptidecomprising SEQ ID NO:36 or SEQ ID NO:37. In some embodiments, theWnt-binding agent comprises a first polypeptide consisting essentiallyof SEQ ID NO:20 and a second polypeptide consisting essentially of SEQID NO:36 or SEQ ID NO:37. In some embodiments, the Wnt-binding agentcomprises a first polypeptide comprising SEQ ID NO:30 and a secondpolypeptide comprising SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, or SEQ ID NO:38. In some embodiments, the Wnt-binding agentcomprises a first polypeptide comprising SEQ ID NO:30 and a secondpolypeptide comprising SEQ ID NO:36 or SEQ ID NO:37. In someembodiments, the Wnt-binding agent comprises a first polypeptidecomprising SEQ ID NO:33 and a second polypeptide comprising SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38. Insome embodiments, the Wnt-binding agent comprises a first polypeptidecomprising SEQ ID NO:33 and a second polypeptide comprising SEQ IDNO:36, SEQ ID NO:37, or SEQ ID NO:35. In some embodiments, theWnt-binding agent comprises a first polypeptide consisting essentiallyof SEQ ID NO:33 and a second polypeptide consisting essentially of SEQID NO:36, SEQ ID NO:37, or SEQ ID NO:35.

In some embodiments, the Wnt-binding agent comprises a first polypeptidecomprising SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, or SEQ ID NO:33; and a second polypeptide comprising SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38,wherein the first polypeptide is connected to the second polypeptide bya linker. In some embodiments, the Wnt-binding agent comprises a firstpolypeptide comprising SEQ ID NO:20 and a second polypeptide comprisingSEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.In some embodiments, the Wnt-binding agent comprises a first polypeptidecomprising SEQ ID NO:20 and a second polypeptide comprising SEQ ID NO:36or SEQ ID NO:37. In some embodiments, the Wnt-binding agent comprises afirst polypeptide consisting essentially of SEQ ID NO:20 and a secondpolypeptide consisting essentially of SEQ ID NO:36 or SEQ ID NO:37. Insome embodiments, the Wnt-binding agent comprises a first polypeptidecomprising SEQ ID NO:30 and a second polypeptide comprising SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38. Insome embodiments, the Wnt-binding agent comprises a first polypeptidecomprising SEQ ID NO:33 and a second polypeptide comprising SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38. Insome embodiments, the Wnt-binding agent comprises a first polypeptidecomprising SEQ ID NO:33 and a second polypeptide comprising SEQ IDNO:36, SEQ ID NO:37, or SEQ ID NO:35. In some embodiments, theWnt-binding agent comprises a first polypeptide consisting essentiallyof SEQ ID NO:33 and a second polypeptide consisting essentially of SEQID NO:36, SEQ ID NO:37, or SEQ ID NO:35.

In some embodiments, the Wnt-binding agent comprises a first polypeptidethat is at least 95% identical to SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33; and a secondpolypeptide comprising SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, or SEQ ID NO:38, wherein the first polypeptide is directly linkedto the second polypeptide. In some embodiments, the Wnt-binding agentcomprises a first polypeptide that is at least 95% identical to SEQ IDNO:20 and a second polypeptide comprising SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38. In some embodiments, theWnt-binding agent comprises a first polypeptide that is at least 95%identical to SEQ ID NO:30 and a second polypeptide comprising SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38. Insome embodiments, the Wnt-binding agent comprises a first polypeptidethat is at least 95% identical to SEQ ID NO:33 and a second polypeptidecomprising SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, orSEQ ID NO:38.

In some embodiments, the Wnt-binding agent comprises a first polypeptidethat is at least 95% identical to SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33; and a secondpolypeptide comprising SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, or SEQ ID NO:38, wherein the first polypeptide is connected tothe second polypeptide by a linker. In some embodiments, the Wnt-bindingagent comprises a first polypeptide that is at least 95% identical toSEQ ID NO:20 and a second polypeptide comprising SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38. In some embodiments,the Wnt-binding agent comprises a first polypeptide that is at least 95%identical to SEQ ID NO:30 and a second polypeptide comprising SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38. Insome embodiments, the Wnt-binding agent comprises a first polypeptidethat is at least 95% identical to SEQ ID NO:33 and a second polypeptidecomprising SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, orSEQ ID NO:38.

FZD proteins contain a signal sequence that directs the transport of theproteins. Signal sequences (also referred to as signal peptides orleader sequences) are located at the N-terminus of nascent polypeptides.They target the polypeptide to the endoplasmic reticulum and theproteins are sorted to their destinations, for example, to the innerspace of an organelle, to an interior membrane, to the cell outermembrane, or to the cell exterior via secretion. Most signal sequencesare cleaved from the protein by a signal peptidase after the proteinsare transported to the endoplasmic reticulum. The cleavage of the signalsequence from the polypeptide usually occurs at a specific site in theamino acid sequence and is dependent upon amino acid residues within thesignal sequence. Although there is usually one specific cleavage site,more than one cleavage site may be recognized and/or used by a signalpeptidase resulting in a non-homogenous N-terminus of the polypeptide.For example, the use of different cleavage sites within a signalsequence can result in a polypeptide expressed with different N-terminalamino acids. Accordingly, in some embodiments, the polypeptidesdescribed herein may comprise a mixture of polypeptides with differentN-termini. In some embodiments, the N-termini differ in length by 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, theN-termini differ in length by 1, 2, 3, 4, or 5 amino acids. In someembodiments, the polypeptide is substantially homogeneous, i.e., thepolypeptides have the same N-terminus. In some embodiments, the signalsequence of the polypeptide comprises one or more (e.g., one, two,three, four, five, six, seven, eight, nine, ten, etc.) amino acidsubstitutions and/or deletions. In some embodiments, the signal sequenceof the polypeptide comprises amino acid substitutions and/or deletionsthat allow one cleavage site to be dominant, thereby resulting in asubstantially homogeneous polypeptide with one N-terminus.

In some embodiments, the Wnt-binding agent comprises an amino acidsequence selected from the group consisting of: SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and SEQID NO:45.

In certain embodiments, the Wnt-binding agent comprises the sequence ofSEQ ID NO:39. In certain embodiments, the agent comprises the sequenceof SEQ ID NO:39, comprising one or more (e.g., one, two, three, four,five, six, seven, eight, nine, ten, etc.) conservative substitutions. Incertain embodiments, the agent comprises a sequence having at leastabout 90%, about 95%, or about 98% sequence identity with SEQ ID NO:39.In certain embodiments, the variants of SEQ ID NO:39 maintain theability to bind one or more human Wnt proteins.

In certain embodiments, the Wnt-binding agent comprises the sequence ofSEQ ID NO:40. In some embodiments, the Wnt-binding agent is SEQ IDNO:40. In certain alternative embodiments, the agent comprises thesequence of SEQ ID NO:40, comprising one or more (e.g., one, two, three,four, five, six, seven, eight, nine, ten, etc.) conservativesubstitutions. In certain embodiments, the agent comprises a sequencehaving at least about 90%, about 95%, or about 98% sequence identitywith SEQ ID NO:40. In certain embodiments, the variants of SEQ ID NO:40maintain the ability to bind one or more human Wnt proteins.

In certain embodiments, the Wnt-binding agent comprises the sequence ofSEQ ID NO:41. In some embodiments, the Wnt-binding agent is SEQ IDNO:41. In certain alternative embodiments, the agent comprises thesequence of SEQ ID NO:41, comprising one or more (e.g., one, two, three,four, five, six, seven, eight, nine, ten, etc.) conservativesubstitutions. In certain embodiments, the agent comprises a sequencehaving at least about 90%, about 95%, or about 98% sequence identitywith SEQ ID NO:41. In certain embodiments, the variants of SEQ ID NO:41maintain the ability to bind one or more human Wnt proteins.

In some embodiments, the Wnt-binding agent is OMP-54F28. In someembodiments, the Wnt-binding agent is not OMP-54F28.

In certain embodiments, a Wnt-binding agent is a polypeptide comprisingan amino acid sequence selected from the group consisting of: SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, and SEQ ID NO:45. In certain embodiments, the polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41. In some embodiments, apolypeptide consists essentially of an amino acid sequence selected fromthe group consisting of: SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.In certain embodiments, the polypeptide comprises the amino acidsequence of SEQ ID NO:39. In some embodiments, the polypeptide comprisesthe amino acid sequence of SEQ ID NO:40. In certain embodiments, thepolypeptide comprises the amino acid sequence of SEQ ID NO:41. Incertain embodiments, the polypeptide comprises the amino acid sequenceof SEQ ID NO:42. In certain embodiments, the polypeptide comprises theamino acid sequence of SEQ ID NO:43. In certain embodiments, thepolypeptide comprises the amino acid sequence of SEQ ID NO:44. Incertain embodiments, the polypeptide comprises the amino acid sequenceof SEQ ID NO:45.

In some embodiments, the polypeptide is a substantially purifiedpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41. In someembodiments, the polypeptide is a substantially purified polypeptidecomprising SEQ ID NO:41. In certain embodiments, the substantiallypurified polypeptide consists of at least 90% of a polypeptide that hasan N-terminal sequence of ASA. In some embodiments, the nascentpolypeptide comprises a signal sequence that results in a substantiallyhomogeneous polypeptide product with one N-terminal sequence.

In certain embodiments, a Wnt-binding agent comprises a Fc region of animmunoglobulin. Those skilled in the art will appreciate that some ofthe binding agents of this invention will comprise fusion proteins inwhich at least a portion of the Fc region has been deleted or otherwisealtered so as to provide desired biochemical characteristics, such asincreased cancer cell localization, increased tumor penetration, reducedserum half-life, or increased serum half-life, when compared with afusion protein of approximately the same immunogenicity comprising anative or unaltered constant region. Modifications to the Fc region mayinclude additions, deletions, or substitutions of one or more aminoacids in one or more domains. The modified fusion proteins disclosedherein may comprise alterations or modifications to one or more of thetwo heavy chain constant domains (CH2 or CH3) or to the hinge region. Inother embodiments, the entire CH2 domain may be removed (ΔCH2constructs). In some embodiments, the omitted constant region domain isreplaced by a short amino acid spacer (e.g., 10 aa residues) thatprovides some of the molecular flexibility typically imparted by theabsent constant region domain.

In some embodiments, the modified fusion proteins are engineered to linkthe CH3 domain directly to the hinge region. In other embodiments, apeptide spacer is inserted between the hinge region and the modified CH2and/or CH3 domains. For example, constructs may be expressed wherein theCH2 domain has been deleted and the remaining CH3 domain (modified orunmodified) is joined to the hinge region with a 5-20 amino acid spacer.Such a spacer may be added to ensure that the regulatory elements of theconstant domain remain free and accessible or that the hinge regionremains flexible. However, it should be noted that amino acid spacersmay, in some cases, prove to be immunogenic and elicit an unwantedimmune response against the construct. Accordingly, in certainembodiments, any spacer added to the construct will be relativelynon-immunogenic so as to maintain the desired biological qualities ofthe fusion protein.

In some embodiments, the modified fusion proteins may have only apartial deletion of a constant domain or substitution of a few or even asingle amino acid. For example, the mutation of a single amino acid inselected areas of the CH2 domain may be enough to substantially reduceFc binding and thereby increase cancer cell localization and/or tumorpenetration. Similarly, it may be desirable to simply delete that partof one or more constant region domains that control a specific effectorfunction (e.g., complement C1q binding). Such partial deletions of theconstant regions may improve selected characteristics of the bindingagent (e.g., serum half-life) while leaving other desirable functionsassociated with the subject constant region domain intact. Moreover, asalluded to above, the constant regions of the disclosed fusion proteinsmay be modified through the mutation or substitution of one or moreamino acids that enhances the profile of the resulting construct. Inthis respect it may be possible to disrupt the activity provided by aconserved binding site (e.g., Fc binding) while substantiallymaintaining the configuration and immunogenic profile of the modifiedfusion protein. In certain embodiments, the modified fusion proteinscomprise the addition of one or more amino acids to the constant regionto enhance desirable characteristics such as decreasing or increasingeffector function, or provide for more cytotoxin or carbohydrateattachment sites.

It is known in the art that the constant region mediates severaleffector functions. For example, binding of the C1 component ofcomplement to the Fc region of IgG or IgM antibodies (bound to antigen)activates the complement system. Activation of complement is importantin the opsonization and lysis of cell pathogens. The activation ofcomplement also stimulates the inflammatory response and can also beinvolved in autoimmune hypersensitivity. In addition, the Fc region ofan immunoglobulin can bind to a cell expressing a Fc receptor (FcR).There are a number of Fc receptors which are specific for differentclasses of antibody, including IgG (gamma receptors), IgE (epsilonreceptors), IgA (alpha receptors) and IgM (mu receptors). Binding ofantibody to Fc receptors on cell surfaces triggers a number of importantand diverse biological responses including engulfment and destruction ofantibody-coated particles, clearance of immune complexes, lysis ofantibody-coated target cells by killer cells, release of inflammatorymediators, placental transfer, and control of immunoglobulin production.

In some embodiments, the modified fusion proteins provide for alteredeffector functions that, in turn, affect the biological profile of theadministered agent. For example, in some embodiments, the deletion orinactivation (through point mutations or other means) of a constantregion domain may reduce Fc receptor binding of the circulating modifiedagent, thereby increasing cancer cell localization and/or tumorpenetration. In other embodiments, the constant region modificationsincrease or reduce the serum half-life of the agent. In someembodiments, the constant region is modified to eliminate disulfidelinkages or oligosaccharide moieties.

In certain embodiments, a modified fusion protein does not have one ormore effector functions normally associated with an Fc region. In someembodiments, the agent has no antibody-dependent cell-mediatedcytotoxicity (ADCC) activity, and/or no complement-dependentcytotoxicity (CDC) activity. In certain embodiments, the agent does notbind to the Fc receptor and/or complement factors. In certainembodiments, the agent has no effector function.

In some embodiments, the Wnt-binding agent (e.g., a soluble receptor)described herein is modified to reduce immunogenicity. In general,immune responses against completely normal human proteins are rare whenthese proteins are used as therapeutics. However, although many fusionproteins comprise polypeptides sequences that are the same as thesequences found in nature, several therapeutic fusion proteins have beenshown to be immunogenic in mammals. In some studies, a fusion proteincomprising a linker has been found to be more immunogenic than a fusionprotein that does not contain a linker. Accordingly, in someembodiments, the polypeptides of the invention are analyzed bycomputation methods to predict immunogenicity. In some embodiments, thepolypeptides are analyzed for the presence of T-cell and/or B-cellepitopes. If any T-cell or B-cell epitopes are identified and/orpredicted, modifications to these regions (e.g., amino acidsubstitutions) may be made to disrupt or destroy the epitopes. Variousalgorithms and software that can be used to predict T-cell and/or B-cellepitopes are known in the art. For example, the software programsSYFPEITHI, HLA Bind, PEPVAC, RANKPEP, DiscoTope, ElliPro, and AntibodyEpitope Prediction are all publicly available.

In some embodiments, a cell producing any of the Wnt-binding agents(e.g., soluble receptors) or polypeptides described herein is provided.In some embodiments, a composition comprising any of the Wnt-bindingagents (e.g., soluble receptors) or polypeptides described herein isprovided. In some embodiments, the composition comprises a polypeptidewherein at least 80%, 90%, 95%, 97%, 98%, or 99% of the polypeptide hasan N-terminal sequence of ASA. In some embodiments, the compositioncomprises a polypeptide wherein 100% of the polypeptide has anN-terminal sequence of ASA. In some embodiments, the compositioncomprises a polypeptide wherein at least 80% of the polypeptide has anN-terminal sequence of ASA. In some embodiments, the compositioncomprises a polypeptide wherein at least 90% of the polypeptide has anN-terminal sequence of ASA. In some embodiments, the compositioncomprises a polypeptide wherein at least 95% of the polypeptide has anN-terminal sequence of ASA.

The polypeptides described herein can be recombinant polypeptides,natural polypeptides, or synthetic polypeptides. It will be recognizedin the art that some amino acid sequences of the invention can be variedwithout significant effect on the structure or function of the protein.If such differences in sequence are contemplated, it should beremembered that there will be critical areas on the protein whichdetermine activity. Thus, the invention further includes variations ofthe polypeptides which show substantial activity or which includeregions of FZD proteins, such as the protein portions discussed herein.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above. Incertain embodiments, the number of substitutions for any given solublereceptor polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5or 3.

Fragments or portions of the polypeptides of the present invention canbe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments can be employed asintermediates for producing the full-length polypeptides. Thesefragments or portion of the polypeptides can also be referred to as“protein fragments” or “polypeptide fragments”. 1001521A “proteinfragment” of this invention is a portion or all of a protein which iscapable of binding to one or more human Wnt proteins or one or morehuman FZD proteins. In some embodiments, the fragment has a highaffinity for one or more human Wnt proteins. In some embodiments, thefragment has a high affinity for one or more human FZD proteins. Somefragments of Wnt-binding agents described herein are protein fragmentscomprising at least part of the extracellular portion of a FZD proteinlinked to at least part of a constant region of an immunoglobulin (e.g.,a Fc region). The binding affinity of the protein fragment can be in therange of about 10⁻¹¹ to 10⁻¹² M, although the affinity can varyconsiderably with fragments of different sizes, ranging from 10⁻⁷ to10⁻¹³ M. In some embodiments, the fragment is about 100 to about 200amino acids in length and comprises a binding domain linked to at leastpart of a constant region of an immunoglobulin.

In some embodiments, the Wnt pathway inhibitors are polyclonalantibodies. Polyclonal antibodies can be prepared by any known method.In some embodiments, polyclonal antibodies are raised by immunizing ananimal (e.g., a rabbit, rat, mouse, goat, donkey) by multiplesubcutaneous or intraperitoneal injections of an antigen of interest(e.g., a purified peptide fragment, full-length recombinant protein, orfusion protein). The antigen can be optionally conjugated to a carriersuch as keyhole limpet hemocyanin (KLH) or serum albumin. The antigen(with or without a carrier protein) is diluted in sterile saline andusually combined with an adjuvant (e.g., Complete or Incomplete Freund'sAdjuvant) to form a stable emulsion. After a sufficient period of time,polyclonal antibodies are recovered from blood and/or ascites of theimmunized animal. The polyclonal antibodies can be purified from serumor ascites according to standard methods in the art including, but notlimited to, affinity chromatography, ion-exchange chromatography, gelelectrophoresis, and dialysis.

In some embodiments, the Wnt pathway inhibitors are monoclonalantibodies. Monoclonal antibodies can be prepared using hybridomamethods known to one of skill in the art (see e.g., Kohler and Milstein,1975, Nature, 256:495-497). In some embodiments, using the hybridomamethod, a mouse, hamster, or other appropriate host animal, is immunizedas described above to elicit from lymphocytes the production ofantibodies that will specifically bind the immunizing antigen. In someembodiments, lymphocytes can be immunized in vitro. In some embodiments,the immunizing antigen can be a human protein or a portion thereof. Insome embodiments, the immunizing antigen can be a mouse protein or aportion thereof.

Following immunization, lymphocytes are isolated and fused with asuitable myeloma cell line using, for example, polyethylene glycol, toform hybridoma cells that can then be selected away from unfusedlymphocytes and myeloma cells. Hybridomas that produce monoclonalantibodies directed specifically against a chosen antigen may beidentified by a variety of methods including, but not limited to,immunoprecipitation, immunoblotting, and in vitro binding assay (e.g.,flow cytometry, FACS, ELISA, and radioimmunoassay). The hybridomas canbe propagated either in in vitro culture using standard methods (J. W.Goding, 1996, Monoclonal Antibodies: Principles and Practice, 3rdEdition, Academic Press, San Diego, Calif.) or in vivo as ascites tumorsin an animal. The monoclonal antibodies can be purified from the culturemedium or ascites fluid according to standard methods in the artincluding, but not limited to, affinity chromatography, ion-exchangechromatography, gel electrophoresis, and dialysis.

In certain embodiments, monoclonal antibodies can be made usingrecombinant DNA techniques as known to one skilled in the art. Thepolynucleotides encoding a monoclonal antibody are isolated from matureB-cells or hybridoma cells, such as by RT-PCR using oligonucleotideprimers that specifically amplify the genes encoding the heavy and lightchains of the antibody, and their sequence is determined usingconventional techniques. The isolated polynucleotides encoding the heavyand light chains are then cloned into suitable expression vectors whichproduce the monoclonal antibodies when transfected into host cells suchas E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin proteins. Inother embodiments, recombinant monoclonal antibodies, or fragmentsthereof, can be isolated from phage display libraries (see e.g.,McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991,Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted for those regions of, forexample, a human antibody to generate a chimeric antibody, or for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the Wnt pathway inhibitor is a humanized antibody.Typically, humanized antibodies are human immunoglobulins in whichresidues from the CDRs are replaced by residues from a CDR of anon-human species (e.g., mouse, rat, rabbit, hamster, etc.) that havethe desired specificity, affinity, and/or binding capability usingmethods known to one skilled in the art. In some embodiments, the Fvframework region residues of a human immunoglobulin are replaced withthe corresponding residues in an antibody from a non-human species thathas the desired specificity, affinity, and/or binding capability. Insome embodiments, the humanized antibody can be further modified by thesubstitution of additional residues either in the Fv framework regionand/or within the replaced non-human residues to refine and optimizeantibody specificity, affinity, and/or capability. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domain regions containing all, or substantiallyall, of the CDRs that correspond to the non-human immunoglobulin whereasall, or substantially all, of the framework regions are those of a humanimmunoglobulin consensus sequence. In some embodiments, the humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region or domain (Fc), typically that of a humanimmunoglobulin. In certain embodiments, such humanized antibodies areused therapeutically because they may reduce antigenicity and HAMA(human anti-mouse antibody) responses when administered to a humansubject.

In certain embodiments, the Wnt pathway inhibitor is a human antibody.Human antibodies can be directly prepared using various techniques knownin the art. In some embodiments, immortalized human B lymphocytesimmunized in vitro or isolated from an immunized individual thatproduces an antibody directed against a target antigen can be generated(see, e.g., Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, p. 77; Boerner et al., 1991, J. Immunol., 147:86-95; andU.S. Pat. Nos. 5,750,373; 5,567,610; and 5,229,275). In someembodiments, the human antibody can be selected from a phage library,where that phage library expresses human antibodies (Vaughan et al.,1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS,95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Markset al., 1991, J. Mol. Biol., 222:581). Alternatively, phage displaytechnology can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. Techniques for the generation and use ofantibody phage libraries are described in U.S. Pat. Nos. 5,969,108;6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., 2008, J. Mol. Bio., 376:1182-1200. Affinity maturation strategiesincluding, but not limited to, chain shuffling (Marks et al., 1992,Bio/Technology, 10:779-783) and site-directed mutagenesis, are known inthe art and may be employed to generate high affinity human antibodies.

In some embodiments, human antibodies can be made in transgenic micethat contain human immunoglobulin loci. These mice are capable, uponimmunization, of producing the full repertoire of human antibodies inthe absence of endogenous immunoglobulin production. This approach isdescribed in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016.

This invention also encompasses bispecific antibodies that specificallyrecognize at least one human FZD protein or at least one Wnt protein.Bispecific antibodies are capable of specifically recognizing andbinding at least two different epitopes. The different epitopes caneither be within the same molecule (e.g., two different epitopes onhuman FZD5) or on different molecules (e.g., one epitope on FZD5 and adifferent epitope on a second protein). In some embodiments, thebispecific antibodies are monoclonal human or humanized antibodies. Insome embodiments, the antibodies can specifically recognize and bind afirst antigen target, (e.g., a FZD protein) as well as a second antigentarget, such as an effector molecule on a leukocyte (e.g., CD2, CD3,CD28, CD80, or CD86) or a Fc receptor (e.g., CD64, CD32, or CD16) so asto focus cellular defense mechanisms to the cell expressing the firstantigen target. In some embodiments, the antibodies can be used todirect cytotoxic agents to cells which express a particular targetantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA.

Techniques for making bispecific antibodies are known by those skilledin the art, see for example, Millstein et al., 1983, Nature,305:537-539; Brennan et al., 1985, Science, 229:81; Suresh et al., 1986,Methods in Enzymol., 121:120; Traunecker et al., 1991, EMBO J.,10:3655-3659; Shalaby et al., 1992, J. Exp. Med., 175:217-225; Kostelnyet al., 1992, J. Immunol., 148:1547-1553; Gruber et al., 1994, J.Immunol., 152:5368; U.S. Pat. No. 5,731,168; and U.S. Patent PublicationNo. 2011/0123532. Bispecific antibodies can be intact antibodies orantibody fragments. Antibodies with more than two valencies are alsocontemplated. For example, trispecific antibodies can be prepared (Tuttet al., 1991, J. Immunol., 147:60). Thus, in certain embodiments theantibodies are multispecific.

In certain embodiments, the antibodies (or other polypeptides) describedherein may be monospecific. For example, in certain embodiments, each ofthe one or more antigen-binding sites that an antibody contains iscapable of binding (or binds) a homologous epitope on differentproteins. In certain embodiments, an antigen-binding site of amonospecific antibody described herein is capable of binding (or binds),for example, FZD5 and FZD7 (i.e., the same epitope is found on both FZD5and FZD7 proteins).

In certain embodiments, the Wnt pathway inhibitor is an antibodyfragment comprising an antigen-binding site. Antibody fragments may havedifferent functions or capabilities than intact antibodies; for example,antibody fragments can have increased tumor penetration. Varioustechniques are known for the production of antibody fragments including,but not limited to, proteolytic digestion of intact antibodies. In someembodiments, antibody fragments include a F(ab′)2 fragment produced bypepsin digestion of an antibody molecule. In some embodiments, antibodyfragments include a Fab fragment generated by reducing the disulfidebridges of an F(ab′)2 fragment. In other embodiments, antibody fragmentsinclude a Fab fragment generated by the treatment of the antibodymolecule with papain and a reducing agent. In certain embodiments,antibody fragments are produced recombinantly. In some embodiments,antibody fragments include Fv or single chain Fv (scFv) fragments. Fab,Fv, and scFv antibody fragments can be expressed in and secreted from E.coli or other host cells, allowing for the production of large amountsof these fragments. In some embodiments, antibody fragments are isolatedfrom antibody phage libraries as discussed herein. For example, methodscan be used for the construction of Fab expression libraries (Huse etal., 1989, Science, 246:1275-1281) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor a FZD or Wnt protein or derivatives, fragments, analogs or homologsthereof. In some embodiments, antibody fragments are linear antibodyfragments. In certain embodiments, antibody fragments are monospecificor bispecific. In certain embodiments, the Wnt pathway inhibitor is ascFv. Various techniques can be used for the production of single-chainantibodies specific to one or more human FZD proteins or one or morehuman Wnt proteins.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis). In some embodiments, an antibody is modified to decrease itsserum half-life.

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It isalso contemplated that the heteroconjugate antibodies can be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins can beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the target (i.e., ahuman FZD protein or a human Wnt protein). In this regard, the variableregion may comprise or be derived from any type of mammal that can beinduced to mount a humoral response and generate immunoglobulins againstthe desired tumor-associated antigen. As such, the variable region ofthe modified antibodies can be, for example, of human, murine, non-humanprimate (e.g. cynomolgus monkeys, macaques, etc.) or rabbit origin. Insome embodiments, both the variable and constant regions of the modifiedimmunoglobulins are human. In other embodiments, the variable regions ofcompatible antibodies (usually derived from a non-human source) can beengineered or specifically tailored to improve the binding properties orreduce the immunogenicity of the molecule. In this respect, variableregions useful in the present invention can be humanized or otherwisealtered through the inclusion of imported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencemodification and/or alteration. Although the CDRs may be derived from anantibody of the same class or even subclass as the antibody from whichthe framework regions are derived, it is envisaged that the CDRs will bederived preferably from an antibody from a different species. It may notbe necessary to replace all of the CDRs with all of the CDRs from thedonor variable region to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of theantigen-binding site.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g., full-length antibodies or immunoreactivefragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics such as increased tumorlocalization and/or increased serum half-life when compared with anantibody of approximately the same immunogenicity comprising a native orunaltered constant region. In some embodiments, the constant region ofthe modified antibodies will comprise a human constant region.Modifications to the constant region compatible with this inventioncomprise additions, deletions or substitutions of one or more aminoacids in one or more domains. The modified antibodies disclosed hereinmay comprise alterations or modifications to one or more of the threeheavy chain constant domains (CH1, CH2 or CH3) and/or to the light chainconstant domain (CL). In some embodiments, one or more domains arepartially or entirely deleted from the constant regions of the modifiedantibodies. In some embodiments, the modified antibodies will comprisedomain deleted constructs or variants wherein the entire CH2 domain hasbeen removed (ACH2 constructs). In some embodiments, the omittedconstant region domain is replaced by a short amino acid spacer (e.g.,10 amino acid residues) that provides some of the molecular flexibilitytypically imparted by the absent constant region.

In some embodiments, the modified antibodies are engineered to fuse theCH3 domain directly to the hinge region of the antibody. In otherembodiments, a peptide spacer is inserted between the hinge region andthe modified CH2 and/or CH3 domains. For example, constructs may beexpressed wherein the CH2 domain has been deleted and the remaining CH3domain (modified or unmodified) is joined to the hinge region with a5-20 amino acid spacer. Such a spacer may be added to ensure that theregulatory elements of the constant domain remain free and accessible orthat the hinge region remains flexible. However, it should be noted thatamino acid spacers may, in some cases, prove to be immunogenic andelicit an unwanted immune response against the construct. Accordingly,in certain embodiments, any spacer added to the construct will berelatively non-immunogenic so as to maintain the desired biologicalqualities of the modified antibodies.

In some embodiments, the modified antibodies may have only a partialdeletion of a constant domain or substitution of a few or even a singleamino acid. For example, the mutation of a single amino acid in selectedareas of the CH2 domain may be enough to substantially reduce Fc bindingand thereby increase cancer cell localization and/or tumor penetration.Similarly, it may be desirable to simply delete the part of one or moreconstant region domains that control a specific effector function (e.g.complement Clq binding). Such partial deletions of the constant regionsmay improve selected characteristics of the antibody (serum half-life)while leaving other desirable functions associated with the subjectconstant region domain intact. Moreover, as alluded to above, theconstant regions of the disclosed antibodies may be modified through themutation or substitution of one or more amino acids that enhances theprofile of the resulting construct. In this respect it may be possibleto disrupt the activity provided by a conserved binding site (e.g., Fcbinding) while substantially maintaining the configuration andimmunogenic profile of the modified antibody. In certain embodiments,the modified antibodies comprise the addition of one or more amino acidsto the constant region to enhance desirable characteristics such asdecreasing or increasing effector function or provide for more cytotoxinor carbohydrate attachment sites.

It is known in the art that the constant region mediates severaleffector functions. For example, binding of the C1 component ofcomplement to the Fc region of IgG or IgM antibodies (bound to antigen)activates the complement system. Activation of complement is importantin the opsonization and lysis of cell pathogens. The activation ofcomplement also stimulates the inflammatory response and can also beinvolved in autoimmune hypersensitivity. In addition, the Fc region ofan antibody can bind a cell expressing a Fc receptor (FcR). There are anumber of Fc receptors which are specific for different classes ofantibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA(alpha receptors) and IgM (mu receptors). Binding of antibody to Fcreceptors on cell surfaces triggers a number of important and diversebiological responses including engulfment and destruction ofantibody-coated particles, clearance of immune complexes, lysis ofantibody-coated target cells by killer cells, release of inflammatorymediators, placental transfer, and control of immunoglobulin production.

In certain embodiments, the Wnt pathway inhibitors are antibodies thatprovide for altered effector functions. These altered effector functionsmay affect the biological profile of the administered antibody. Forexample, in some embodiments, the deletion or inactivation (throughpoint mutations or other means) of a constant region domain may reduceFc receptor binding of the circulating modified antibody (e.g., anti-FZDantibody) thereby increasing cancer cell localization and/or tumorpenetration. In other embodiments, the constant region modificationsincrease or reduce the serum half-life of the antibody. In someembodiments, the constant region is modified to eliminate disulfidelinkages or oligosaccharide moieties. Modifications to the constantregion in accordance with this invention may easily be made using wellknown biochemical or molecular engineering techniques well within thepurview of the skilled artisan.

In certain embodiments, a Wnt pathway inhibitor is an antibody does nothave one or more effector functions. For instance, in some embodiments,the antibody has no ADCC activity, and/or no CDC activity. In certainembodiments, the antibody does not bind an Fc receptor, and/orcomplement factors. In certain embodiments, the antibody has no effectorfunction.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized, and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art and described herein.

Thus, the present invention provides methods for producing an antibody.In some embodiments, the method for producing an antibody comprisesusing hybridoma techniques. In some embodiments, a method for producingan antibody that binds a human FZD protein is provided. In someembodiments, a method for producing an antibody that binds a human Wntprotein is provided. In some embodiments, the method of generating anantibody comprises screening a human phage library. In some embodiments,the antibody is identified using a membrane-bound heterodimeric moleculecomprising a single antigen-binding site. In some non-limitingembodiments, the antibody is identified using methods and polypeptidesdescribed in U.S. Patent Publication No. 2011/0287979.

The present invention further provides methods of identifying anantibody that binds at least one FZD protein. In some embodiments, theantibody is identified by screening by FACS for binding to a FZD proteinor a portion thereof. In some embodiments, the antibody is identified byscreening using ELISA for binding to a FZD protein. In some embodiments,the antibody is identified by screening by FACS for blocking of bindingof a FZD protein to a human Wnt protein. In some embodiments, theantibody is identified by screening for inhibition or blocking of Wntpathway signaling.

The present invention further provides methods of identifying anantibody that binds at least one Wnt protein. In some embodiments, theantibody is identified by screening by FACS for binding to a Wnt proteinor a portion thereof. In some embodiments, the antibody is identified byscreening using ELISA for binding to a Wnt protein. In some embodiments,the antibody is identified by screening by FACS for blocking of bindingof a Wnt protein to a human FZD protein. In some embodiments, theantibody is identified by screening for inhibition or blocking of Wntpathway signaling.

In some embodiments, a method of generating an antibody to at least onehuman FZD protein comprises screening an antibody-expressing library forantibodies that bind a human FZD protein. In some embodiments, theantibody-expressing library is a phage library. In some embodiments, theantibody-expressing library is a mammalian cell library. In someembodiments, the screening comprises panning. In some embodiments,antibodies identified in a first screening, are screened again using adifferent FZD protein thereby identifying an antibody that binds thefirst FZD protein and a second FZD protein. In some embodiments, theantibody identified in the screening binds the first FZD protein and atleast one other FZD protein. In certain embodiments, the at least oneother FZD protein is selected from the group consisting of FZD 1, FZD2,FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. In certainembodiments, the antibody identified in the screening binds FZD1, FZD2,FZD5, FZD7, and FZD8. In some embodiments, the antibody identified inthe screening is a FZD antagonist. In some embodiments, the antibodyidentified by the methods described herein inhibits the Wnt pathway. Insome embodiments, the antibody identified in the screening inhibitsβ-catenin signaling.

In some embodiments, a method of generating an antibody to at least onehuman Wnt protein comprises screening an antibody-expressing library forantibodies that bind a human Wnt protein. In some embodiments, theantibody-expressing library is a phage library. In some embodiments, theantibody-expressing library is a mammalian cell library. In someembodiments, the screening comprises panning. In some embodiments,antibodies identified in a first screening, are screened again using adifferent Wnt protein thereby identifying an antibody that binds a firstWnt protein and a second Wnt protein. In some embodiments, the antibodyidentified in the screening binds a first Wnt protein and at least oneother Wnt protein. In certain embodiments, the at least one other FZDprotein is selected from the group consisting of Wnt1, Wnt2, Wnt2b,Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. In someembodiments, the antibody identified in the screening is a Wntantagonist. In some embodiments, the antibody identified by the methodsdescribed herein inhibits the Wnt pathway. In some embodiments, theantibody identified in the screening inhibits β-catenin signaling.

In certain embodiments, the antibodies described herein are isolated. Incertain embodiments, the antibodies described herein are substantiallypure.

In some embodiments of the present invention, the Wnt pathway inhibitorsare polypeptides. The polypeptides can be recombinant polypeptides,natural polypeptides, or synthetic polypeptides comprising an antibody,or fragment thereof, that bind at least one human FZD protein or atleast one Wnt protein. It will be recognized in the art that some aminoacid sequences of the invention can be varied without significant effecton the structure or function of the protein. Thus, the invention furtherincludes variations of the polypeptides which show substantial activityor which include regions of an antibody, or fragment thereof, against ahuman FZD protein or a Wnt protein. In some embodiments, amino acidsequence variations of FZD-binding polypeptides or Wnt-bindingpolypeptides include deletions, insertions, inversions, repeats, and/orother types of substitutions.

The polypeptides, analogs and variants thereof, can be further modifiedto contain additional chemical moieties not normally part of thepolypeptide. The derivatized moieties can improve the solubility, thebiological half-life, and/or absorption of the polypeptide. The moietiescan also reduce or eliminate any undesirable side effects of thepolypeptides and variants. An overview for chemical moieties can befound in Remington: The Science and Practice of Pharmacy, 22^(st)Edition, 2012, Pharmaceutical Press, London.

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthesis methods to constructing a DNA sequence encoding polypeptidesequences and expressing those sequences in a suitable host. In someembodiments, a DNA sequence is constructed using recombinant technologyby isolating or synthesizing a DNA sequence encoding a wild-type proteinof interest. Optionally, the sequence can be mutagenized bysite-specific mutagenesis to provide functional analogs thereof.

In some embodiments, a DNA sequence encoding a polypeptide of interestmay be constructed by chemical synthesis using an oligonucleotidesynthesizer. Oligonucleotides can be designed based on the amino acidsequence of the desired polypeptide and selecting those codons that arefavored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizea polynucleotide sequence encoding an isolated polypeptide of interest.For example, a complete amino acid sequence can be used to construct aback-translated gene. Further, a DNA oligomer containing a nucleotidesequence coding for the particular isolated polypeptide can besynthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis, or anothermethod), the polynucleotide sequences encoding a particular polypeptideof interest can be inserted into an expression vector and operativelylinked to an expression control sequence appropriate for expression ofthe protein in a desired host. Proper assembly can be confirmed bynucleotide sequencing, restriction enzyme mapping, and/or expression ofa biologically active polypeptide in a suitable host. As is well-knownin the art, in order to obtain high expression levels of a transfectedgene in a host, the gene must be operatively linked to transcriptionaland translational expression control sequences that are functional inthe chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding binding agents (e.g., antibodies orsoluble receptors), or fragments thereof, against a human FZD protein ora Wnt protein. For example, recombinant expression vectors can bereplicable DNA constructs which have synthetic or cDNA-derived DNAfragments encoding a polypeptide chain of a FZD-binding agent, aWnt-binding agent, an anti-FZD antibody or fragment thereof, an anti-Wntantibody or fragment thereof, or a FZD-Fc soluble receptor operativelylinked to suitable transcriptional and/or translational regulatoryelements derived from mammalian, microbial, viral or insect genes. Atranscriptional unit generally comprises an assembly of (1) a geneticelement or elements having a regulatory role in gene expression, forexample, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences. Regulatory elements can include an operatorsequence to control transcription. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants can additionally beincorporated. DNA regions are “operatively linked” when they arefunctionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. In some embodiments,structural elements intended for use in yeast expression systems includea leader sequence enabling extracellular secretion of translated proteinby a host cell. In other embodiments, where recombinant protein isexpressed without a leader or transport sequence, it can include anN-terminal methionine residue. This residue can optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

The choice of an expression control sequence and an expression vectordepends upon the choice of host. A wide variety of expressionhost/vector combinations can be employed. Useful expression vectors foreukaryotic hosts include, for example, vectors comprising expressioncontrol sequences from SV40, bovine papilloma virus, adenovirus, andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, including pCR1,pBR322, pMB9 and their derivatives, and wider host range plasmids, suchas M13 and other filamentous single-stranded DNA phages.

Suitable host cells for expression of a FZD-binding or Wnt-binding agent(or a protein to use as an antigen) include prokaryotes, yeast cells,insect cells, or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram-negative orgram-positive organisms, for example E. coli or Bacillus. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems may also be employed.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described by Pouwels etal. (1985, Cloning Vectors: A Laboratory Manual, Elsevier, New York,N.Y.). Additional information regarding methods of protein production,including antibody production, can be found, e.g., in U.S. PatentPublication No. 2008/0187954, U.S. Pat. Nos. 6,413,746 and 6,660,501,and International Patent Publication No. WO 2004/009823.

Various mammalian culture systems are used to express recombinantpolypeptides. Expression of recombinant proteins in mammalian cells maybe preferred because such proteins are generally correctly folded,appropriately modified, and biologically functional. Examples ofsuitable mammalian host cell lines include COS-7 (monkeykidney-derived), L-929 (murine fibroblast-derived), C127 (murine mammarytumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamsterovary-derived), HeLa (human cervical cancer-derived), BHK (hamsterkidney fibroblast-derived), HEK-293 (human embryonic kidney-derived)cell lines and variants thereof. Mammalian expression vectors cancomprise non-transcribed elements such as an origin of replication, asuitable promoter and enhancer linked to the gene to be expressed, andother 5′ or 3′ flanking non-transcribed sequences, and 5′ or 3′non-translated sequences, such as necessary ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences.

Expression of recombinant proteins in insect cell culture systems (e.g.,baculovirus) also offers a robust method for producing correctly foldedand biologically functional proteins. Baculovirus systems for productionof heterologous proteins in insect cells are well-known to those ofskill in the art (see, e.g., Luckow and Summers, 1988, Bio/Technology,6:47).

Thus, the present invention provides cells comprising the FZD-bindingagents or the Wnt-binding agents described herein. In some embodiments,the cells produce the binding agents (e.g., antibodies or solublereceptors) described herein. In certain embodiments, the cells producean antibody. In certain embodiments, the cells produce antibody 18R5. Insome embodiments, the cells produce a soluble receptor. In someembodiments, the cells produce a FZD-Fc soluble receptor. In someembodiments, the cells produce a FZD8-Fc soluble receptor. In someembodiments, the cells produce FZD8-Fc soluble receptor 54F28.

The proteins produced by a transformed host can be purified according toany suitable method. Standard methods include chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for proteinpurification. Affinity tags such as hexa-histidine, maltose bindingdomain, influenza coat sequence, and glutathione-S-transferase can beattached to the protein to allow easy purification by passage over anappropriate affinity column. Isolated proteins can also be physicallycharacterized using such techniques as proteolysis, mass spectrometry(MS), nuclear magnetic resonance (NMR), high performance liquidchromatography (HPLC), and x-ray crystallography.

In some embodiments, supernatants from expression systems which secreterecombinant protein into culture media can be first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a suitablepurification matrix. In some embodiments, an anion exchange resin can beemployed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose, or other types commonly employed in proteinpurification. In some embodiments, a cation exchange step can beemployed. Suitable cation exchangers include various insoluble matricescomprising sulfopropyl or carboxymethyl groups. In some embodiments, ahydroxyapatite media can be employed, including but not limited to,ceramic hydroxyapatite (CHT). In certain embodiments, one or morereverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g.,silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a binding agent. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a homogeneous recombinant protein.

In some embodiments, recombinant protein produced in bacterial culturecan be isolated, for example, by initial extraction from cell pellets,followed by one or more concentration, salting-out, aqueous ionexchange, or size exclusion chromatography steps. HPLC can be employedfor final purification steps. Microbial cells employed in expression ofa recombinant protein can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption, or useof cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNos. 2008/0312425, 2008/0177048, and 2009/0187005.

In certain embodiments, the Wnt-binding agent or the FZD-binding agentis a polypeptide that is not an antibody. A variety of methods foridentifying and producing non-antibody polypeptides that bind with highaffinity to a protein target are known in the art. See, e.g., Skerra,2007, Curr. Opin. Biotechnol., 18:295-304; Hosse et al., 2006, ProteinScience, 15:14-27; Gill et al., 2006, Curr. Opin. Biotechnol.,17:653-658; Nygren, 2008, FEBSJ., 275:2668-76; and Skerra, 2008, FEBSJ.,275:2677-83. In certain embodiments, phage display technology may beused to produce and/or identify a FZD-binding or Wnt-bindingpolypeptide. In certain embodiments, the polypeptide comprises a proteinscaffold of a type selected from the group consisting of protein A,protein G, a lipocalin, a fibronectin domain, an ankyrin consensusrepeat domain, and thioredoxin.

In certain embodiments, the binding agents can be used in any one of anumber of conjugated (i.e. an immunoconjugate or radioconjugate) ornon-conjugated forms. In certain embodiments, antibodies can be used ina non-conjugated form to harness the subject's natural defensemechanisms including complement-dependent cytotoxicity and antibodydependent cellular toxicity to eliminate the malignant or cancer cells.

In some embodiments, the binding agent is conjugated to a cytotoxicagent. In some embodiments, the cytotoxic agent is a chemotherapeuticagent including, but not limited to, methotrexate, adriamicin,doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents. In some embodiments, the cytotoxic agent is anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof, including, but not limited to, diphtheriaA chain, nonbinding active fragments of diphtheria toxin, exotoxin Achain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,Aleurites fordii proteins, dianthin proteins, Phytolaca americanaproteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor,curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin,restrictocin, phenomycin, enomycin, and the tricothecenes. In someembodiments, the cytotoxic agent is a radioisotope to produce aradioconjugate or a radioconjugated antibody. A variety of radionuclidesare available for the production of radioconjugated antibodiesincluding, but not limited to, ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹³¹In,¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re and ²¹²Bi. In someembodiments, conjugates of an antibody and one or more small moleculetoxins, such as a calicheamicin, maytansinoids, a trichothene, andCC1065, and the derivatives of these toxins that have toxin activity,can be produced. In certain embodiments, conjugates of an antibody and acytotoxic agent are made using a variety of bifunctionalprotein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

In certain embodiments, the Wnt pathway inhibitor (e.g., antibody orsoluble receptor) is an antagonist of at least one Wnt protein (i.e., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 Wnt proteins). In certain embodiments, theWnt pathway inhibitor inhibits activity of the Wnt protein(s) to whichit binds. In certain embodiments, the Wnt pathway inhibitor inhibits atleast about 10%, at least about 20%, at least about 30%, at least about50%, at least about 75%, at least about 90%, or about 100% of theactivity of the human Wnt protein(s) to which it binds.

In certain embodiments, the Wnt pathway inhibitor (e.g., antibody orsoluble receptor) inhibits binding of at least one human Wnt to anappropriate receptor. In certain embodiments, the Wnt pathway inhibitorinhibits binding of at least one human Wnt protein to one or more humanFZD proteins. In some embodiments, the at least one Wnt protein isselected from the group consisting of: Wnt1, Wnt2, Wnt2b/13, Wnt3,Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a,Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. In some embodiments, the one ormore human FZD proteins are selected from the group consisting of: FZD1,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD 10. In certainembodiments, the Wnt pathway inhibitor inhibits binding of one or moreWnt proteins to FZD1, FZD2, FZD4, FZD5, FZD7, and/or FZD8. In certainembodiments, the Wnt pathway inhibitor inhibits binding of one or moreWnt proteins to FZD8. In certain embodiments, the inhibition of bindingof a particular Wnt to a FZD protein by a Wnt pathway inhibitor is atleast about 10%, at least about 25%, at least about 50%, at least about75%, at least about 90%, or at least about 95%. In certain embodiments,an agent that inhibits binding of a Wnt to a FZD protein, also inhibitsWnt pathway signaling. In certain embodiments, a Wnt pathway inhibitorthat inhibits human Wnt pathway signaling is an antibody. In certainembodiments, a Wnt pathway inhibitor that inhibits human Wnt pathwaysignaling is a FZD-Fc soluble receptor. In certain embodiments, a Wntpathway inhibitor that inhibits human Wnt pathway signaling is a FZD8-Fcsoluble receptor. In certain embodiments, a Wnt pathway inhibitor thatinhibits human Wnt pathway signaling is soluble receptor 54F28.

In certain embodiments, the Wnt pathway inhibitors (e.g., antibody orsoluble receptor) described herein are antagonists of at least one humanWnt protein and inhibit Wnt activity. In certain embodiments, the Wntpathway inhibitor inhibits Wnt activity by at least about 10%, at leastabout 20%, at least about 30%, at least about 50%, at least about 75%,at least about 90%, or about 100%. In some embodiments, the Wnt pathwayinhibitor inhibits activity of one, two, three, four, five or more Wntproteins. In some embodiments, the Wnt pathway inhibitor inhibitsactivity of at least one human Wnt protein selected from the groupconsisting of: Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6,Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, andWnt16. In some embodiments, the Wnt-binding agent binds at least one Wntprotein selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3,Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. In certainembodiments, the at least one Wnt protein is selected from the groupconsisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, andWnt10b. In certain embodiments, a Wnt pathway inhibitor that inhibitshuman Wnt activity is an antibody. In certain embodiments, a Wnt pathwayinhibitor that inhibits human Wnt activity is a FZD-Fc soluble receptor.In certain embodiments, a Wnt pathway inhibitor that inhibits human Wntactivity is a FZD8-Fc soluble receptor. In certain embodiments, a Wntpathway inhibitor that inhibits human Wnt activity is soluble receptor54F28.

In certain embodiments, the Wnt pathway inhibitor described herein is anantagonist of at least one human FZD protein and inhibits FZD activity.In certain embodiments, the Wnt pathway inhibitor inhibits FZD activityby at least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 75%, at least about 90%, or about 100%. Insome embodiments, the Wnt pathway inhibitor inhibits activity of one,two, three, four, five or more FZD proteins. In some embodiments, theWnt pathway inhibitor inhibits activity of at least one human FZDprotein selected from the group consisting of: FZD1, FZD2, FZD3, FZD4,FZD5, FZD6, FZD7, FZD8, FZD9, and FZD 10. In certain embodiments, theWnt pathway inhibitor inhibits activity of FZD 1, FZD2, FZD4, FZD5,FZD7, and/or FZD8. In certain embodiments, the Wnt pathway inhibitorinhibits activity of FZD8. In some embodiments, the Wnt pathwayinhibitor is an anti-FZD antibody. In certain embodiments, the Wntpathway inhibitor is anti-FZD antibody 18R5.

In certain embodiments, the Wnt pathway inhibitor described herein is anantagonist of at least one human Wnt protein and inhibits Wnt signaling.In certain embodiments, the Wnt pathway inhibitor inhibits Wnt signalingby at least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 75%, at least about 90%, or about 100%. Insome embodiments, the Wnt pathway inhibitor inhibits signaling by one,two, three, four, five or more Wnt proteins. In some embodiments, theWnt pathway inhibitor inhibits signaling of at least one Wnt proteinselected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a,Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. In certain embodiments,a Wnt pathway inhibitor that inhibits Wnt signaling is an antibody. Incertain embodiments, a Wnt pathway inhibitor that inhibits Wnt signalingis a soluble receptor. In certain embodiments, a Wnt pathway inhibitorthat inhibits Wnt signaling is a FZD-Fc soluble receptor. In certainembodiments, a Wnt pathway inhibitor that inhibits Wnt signaling is aFZD8-Fc soluble receptor. In certain embodiments, a Wnt pathwayinhibitor that inhibits Wnt signaling is soluble receptor 54F28.

In certain embodiments, a Wnt pathway inhibitor described herein is anantagonist of β-catenin signaling. In certain embodiments, the Wntpathway inhibitor inhibits β-catenin signaling by at least about 10%, atleast about 20%, at least about 30%, at least about 50%, at least about75%, at least about 90%, or about 100%. In certain embodiments, a Wntpathway inhibitor that inhibits β-catenin signaling is an antibody. Incertain embodiments, a Wnt pathway inhibitor that inhibits β-cateninsignaling is an anti-FZD antibody. In certain embodiments, a Wnt pathwayinhibitor that inhibits β-catenin signaling is antibody 18R5. In certainembodiments, a Wnt pathway inhibitor that inhibits β-catenin signalingis a soluble receptor. In certain embodiments, a Wnt pathway inhibitorthat inhibits β-catenin signaling is a FZD-Fc soluble receptor. Incertain embodiments, a Wnt pathway inhibitor that inhibits β-cateninsignaling is a FZD8-Fc soluble receptor.

In certain embodiments, the Wnt pathway inhibitor described hereininhibits binding of at least one Wnt protein to a receptor. In certainembodiments, the Wnt pathway inhibitor inhibits binding of at least onehuman Wnt protein to one or more of its receptors. In some embodiments,the Wnt pathway inhibitor inhibits binding of at least one Wnt proteinto at least one FZD protein. In some embodiments, the Wnt-binding agentinhibits binding of at least one Wnt protein to FZD 1, FZD2, FZD3, FZD4,FDZ5, FDZ6, FDZ7, FDZ8, FDZ9, and/or FZD10. In certain embodiments, theinhibition of binding of at least one Wnt to at least one FZD protein isat least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 90%, or at least about 95%. In certainembodiments, a Wnt pathway inhibitor that inhibits binding of at leastone Wnt to at least one FZD protein further inhibits Wnt pathwaysignaling and/or β-catenin signaling. In certain embodiments, a Wntpathway inhibitor that inhibits binding of at least one human Wnt to atleast one FZD protein is an antibody. In certain embodiments, a Wntpathway inhibitor that inhibits binding of at least one human Wnt to atleast one FZD protein is an anti-FZD antibody. In certain embodiments, aWnt pathway inhibitor that inhibits binding of at least one human Wnt toat least one FZD protein is antibody 18R5. In certain embodiments, a Wntpathway inhibitor that inhibits binding of at least one human Wnt to atleast one FZD protein is a soluble receptor. In certain embodiments, aWnt pathway inhibitor that inhibits binding of at least one human Wnt toat least one FZD protein is a FZD-Fc soluble receptor. In certainembodiments, a Wnt pathway inhibitor that inhibits binding of at leastone human Wnt to at least one FZD protein is a FZD8-Fc soluble receptor.In certain embodiments, a Wnt pathway inhibitor that inhibits binding ofat least one human Wnt to at least one FZD protein is FZD8-Fc solublereceptor 54F28.

In certain embodiments, the Wnt pathway inhibitor described hereinblocks binding of at least one Wnt to a receptor. In certainembodiments, the Wnt pathway inhibitor blocks binding of at least onehuman Wnt protein to one or more of its receptors. In some embodiments,the Wnt pathway inhibitor blocks binding of at least one Wnt to at leastone FZD protein. In some embodiments, the Wnt pathway inhibitor blocksbinding of at least one Wnt protein to FZD 1, FZD2, FZD3, FZD4, FDZ5,FDZ6, FDZ7, FDZ8, FDZ9, and/or FZD10. In certain embodiments, theblocking of binding of at least one Wnt to at least one FZD protein isat least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 90%, or at least about 95%. In certainembodiments, a Wnt pathway inhibitor that blocks binding of at least oneWnt protein to at least one FZD protein further inhibits Wnt pathwaysignaling and/or β-catenin signaling. In certain embodiments, a Wntpathway inhibitor that blocks binding of at least one human Wnt to atleast one FZD protein is an antibody. In certain embodiments, a Wntpathway inhibitor that blocks binding of at least one human Wnt to atleast one FZD protein is an anti-FZD antibody. In certain embodiments, aWnt pathway inhibitor that blocks binding of at least one human Wnt toat least one FZD protein is antibody 18R5. In certain embodiments, a Wntpathway inhibitor that blocks binding of at least one human Wnt to atleast one FZD protein is a soluble receptor. In certain embodiments, aWnt pathway inhibitor that blocks binding of at least one human Wnt toat least one FZD protein is a FZD-Fc soluble receptor. In certainembodiments, a Wnt pathway inhibitor that blocks binding of at least onehuman Wnt to at least one FZD protein is a FZD8-Fc soluble receptor. Incertain embodiments, a Wnt pathway inhibitor that blocks binding of atleast one human Wnt to at least one FZD protein is soluble receptor54F28.

In certain embodiments, the Wnt pathway inhibitor described hereininhibits Wnt pathway signaling. It is understood that a Wnt pathwayinhibitor that inhibits Wnt pathway signaling may, in certainembodiments, inhibit signaling by one or more receptors in the Wntsignaling pathway but not necessarily inhibit signaling by allreceptors. In certain alternative embodiments, Wnt pathway signaling byall human receptors may be inhibited. In certain embodiments, Wntpathway signaling by one or more receptors selected from the groupconsisting of FZD 1, FZD2, FZD3, FZD4, FDZ5, FDZ6, FDZ7, FDZ8, FDZ9, andFZD10 is inhibited. In certain embodiments, the inhibition of Wntpathway signaling by a Wnt pathway inhibitor is a reduction in the levelof Wnt pathway signaling of at least about 10%, at least about 25%, atleast about 50%, at least about 75%, at least about 90%, or at leastabout 95%. In some embodiments, a Wnt pathway inhibitor that inhibitsWnt pathway signaling is an antibody. In some embodiments, a Wnt pathwayinhibitor that inhibits Wnt pathway signaling is an anti-FZD antibody.In some embodiments, a Wnt pathway inhibitor that inhibits Wnt pathwaysignaling is antibody 18R5. In some embodiments, a Wnt pathway inhibitorthat inhibits Wnt pathway signaling is a soluble receptor. In someembodiments, a Wnt pathway inhibitor that inhibits Wnt pathway signalingis a FZD-Fc soluble receptor. In some embodiments, a Wnt pathwayinhibitor that inhibits Wnt pathway signaling is a FZD8-Fc solublereceptor. In some embodiments, a Wnt pathway inhibitor that inhibits Wntpathway signaling is soluble receptor 54F28.

In certain embodiments, the Wnt pathway inhibitor described hereininhibits activation of β-catenin. It is understood that a Wnt pathwayinhibitor that inhibits activation of β-catenin may, in certainembodiments, inhibit activation of β-catenin by one or more receptors,but not necessarily inhibit activation of β-catenin by all receptors. Incertain alternative embodiments, activation of β-catenin by all humanreceptors may be inhibited. In certain embodiments, activation ofβ-catenin by one or more receptors selected from the group consisting ofFZD1, FZD2, FZD3, FZD4, FDZ5, FDZ6, FDZ7, FDZ8, FDZ9, and FZD10 isinhibited. In certain embodiments, the inhibition of activation ofβ-catenin by a Wnt-binding agent is a reduction in the level ofactivation of β-catenin of at least about 10%, at least about 25%, atleast about 50%, at least about 75%, at least about 90%, or at leastabout 95%. In some embodiments, a Wnt pathway inhibitor that inhibitsactivation of β-catenin is an antibody. In some embodiments, a Wntpathway inhibitor that inhibits activation of β-catenin is an anti-FZDantibody. In some embodiments, a Wnt pathway inhibitor that inhibitsactivation of β-catenin is antibody 18R5. In some embodiments, a Wntpathway inhibitor that inhibits activation of β-catenin is a solublereceptor. In some embodiments, a Wnt pathway inhibitor that inhibitsactivation of β-catenin is a FZD-Fc soluble receptor. In someembodiments, a Wnt pathway inhibitor that inhibits activation ofβ-catenin is a FZD8-Fc soluble receptor. In some embodiments, a Wntpathway inhibitor that inhibits activation of β-catenin is solublereceptor 54F28.

In vivo and in vitro assays for determining whether a Wnt pathwayinhibitor inhibits β-catenin signaling are known in the art. Forexample, cell-based, luciferase reporter assays utilizing a TCF/Lucreporter vector containing multiple copies of the TCF-binding domainupstream of a firefly luciferase reporter gene may be used to measureβ-catenin signaling levels in vitro (Gazit et al., 1999, Oncogene, 18;5959-66; TOPflash, Millipore, Billerica Mass.). The level of β-cateninsignaling in the presence of one or more Wnt proteins (e.g., Wnt(s)expressed by transfected cells or provided by Wnt-conditioned media) inthe presence of a binding agent is compared to the level of signalingwithout the binding agent present. In addition to the TCF/Luc reporterassay, the effect of a binding agent (or candidate agent) on β-cateninsignaling may be measured in vitro or in vivo by measuring the effect ofthe agent on the level of expression of β-catenin-regulated genes, suchas c-myc (He et al., 1998, Science, 281:1509-12), cyclin D1 (Tetsu etal., 1999, Nature, 398:422-6), and/or fibronectin (Gradl et al. 1999,Mol. Cell. Biol., 19:5576-87). In certain embodiments, the effect of abinding agent on β-catenin signaling may also be assessed by measuringthe effect of the agent on the phosphorylation state of Dishevelled-1,Dishevelled-2, Dishevelled-3, LRP5, LRP6, and/or β-catenin.

In certain embodiments, a Wnt pathway inhibitor has one or more of thefollowing effects: inhibit proliferation of tumor cells, inhibit tumorgrowth, reduce the frequency of cancer stem cells in a tumor, reduce thetumorigenicity of a tumor, reduce the tumorigenicity of a tumor byreducing the frequency of cancer stem cells in the tumor, trigger celldeath of tumor cells, induce cells in a tumor to differentiate,differentiate tumorigenic cells to a non-tumorigenic state, induceexpression of differentiation markers in the tumor cells, preventmetastasis of tumor cells, or decrease survival of tumor cells.

In certain embodiments, a Wnt pathway inhibitor is capable of inhibitingtumor growth. In certain embodiments, a Wnt pathway inhibitor is capableof inhibiting tumor growth in vivo (e.g., in a xenograft mouse model,and/or in a human having cancer). In some embodiments, the tumor is atumor selected from the group consisting of colorectal tumor, colontumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breasttumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma,cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. Incertain embodiments, the tumor is melanoma. In certain embodiments, thetumor is a colorectal tumor. In certain embodiments, the tumor is apancreatic tumor. In certain embodiments, the tumor is a breast tumor.In certain embodiments, the tumor is a Wnt-dependent tumor.

In certain embodiments, a Wnt pathway inhibitor is capable of reducingthe tumorigenicity of a tumor. In certain embodiments, a Wnt pathwayinhibitor is capable of reducing the tumorigenicity of a tumorcomprising cancer stem cells in an animal model, such as a mousexenograft model. In certain embodiments, the number or frequency ofcancer stem cells in a tumor is reduced by at least about two-fold,about three-fold, about five-fold, about ten-fold, about 50-fold, about100-fold, or about 1000-fold. In certain embodiments, the reduction inthe number or frequency of cancer stem cells is determined by limitingdilution assay using an animal model. Additional examples and guidanceregarding the use of limiting dilution assays to determine a reductionin the number or frequency of cancer stem cells in a tumor can be found,e.g., in International Publication No. WO 2008/042236, and U.S. PatentPublication Nos. 2008/0064049 and 2008/0178305.

In certain embodiments, the Wnt pathway inhibitors described herein areactive in vivo for at least 1 hour, at least about 2 hours, at leastabout 5 hours, at least about 10 hours, at least about 24 hours, atleast about 2 days, at least about 3 days, at least about 1 week, or atleast about 2 weeks. In certain embodiments, the Wnt pathway inhibitoris an IgG (e.g., IgG1 or IgG2) antibody that is active in vivo for atleast 1 hour, at least about 2 hours, at least about 5 hours, at leastabout 10 hours, at least about 24 hours, at least about 2 days, at leastabout 3 days, at least about 1 week, or at least about 2 weeks. Incertain embodiments, the Wnt pathway inhibitor is a fusion protein thatis active in vivo for at least 1 hour, at least about 2 hours, at leastabout 5 hours, at least about 10 hours, at least about 24 hours, atleast about 2 days, at least about 3 days, at least about 1 week, or atleast about 2 weeks.

In certain embodiments, the Wnt pathway inhibitors described herein havea circulating half-life in mice, cynomolgus monkeys, or humans of atleast about 5 hours, at least about 10 hours, at least about 24 hours,at least about 2 days, at least about 3 days, at least about 1 week, orat least about 2 weeks. In certain embodiments, the Wnt pathwayinhibitor is an IgG (e.g., IgG1 or IgG2) antibody that has a circulatinghalf-life in mice, cynomolgus monkeys, or humans of at least about 5hours, at least about 10 hours, at least about 24 hours, at least about2 days, at least about 3 days, at least about 1 week, or at least about2 weeks. In certain embodiments, the Wnt pathway inhibitor is a fusionprotein that has a circulating half-life in mice, cynomolgus monkeys, orhumans of at least about 5 hours, at least about 10 hours, at leastabout 24 hours, at least about 2 days, at least about 3 days, at leastabout 1 week, or at least about 2 weeks. Methods of increasing (ordecreasing) the half-life of agents such as polypeptides and antibodiesare known in the art. For example, known methods of increasing thecirculating half-life of IgG antibodies include the introduction ofmutations in the Fc region which increase the pH-dependent binding ofthe antibody to the neonatal Fc receptor (FcRn) at pH 6.0 (see, e.g.,U.S. Patent Publication Nos. 2005/0276799, 2007/0148164, and2007/0122403). Known methods of increasing the circulating half-life ofantibody fragments lacking the Fc region include such techniques asPEGylation.

III. METHODS OF USE AND PHARMACEUTICAL COMPOSITIONS

The present invention provides methods of treating diseases such ascancer with a Wnt pathway inhibitor, while screening for, monitoring,reducing, preventing, attenuating, and/or mitigating side effects and/ortoxicities, including, but not limited to skeletal-related side effectsand/or toxicities associated with the Wnt pathway inhibitor. Sideeffects and/or toxicities associated with cancer treatment may include,but are not limited to, fatigue, vomiting, nausea, diarrhea, pain, hairloss, neutropenia, anemia, thrombocytopenia, cardiovascular-relatedcomplications, skeletal-related complications, and any combinationthereof. As used herein, “skeletal-related complications” (e.g.,skeletal-related side effects and/or toxicities) include but are notlimited to, osteopenia, osteoporosis, bone fractures (including silentfractures), and combinations thereof. Thus, in some aspects and/orembodiments of the methods described herein, the screening for,monitoring, reducing, preventing, attenuating, and/or mitigatingskeletal-related side effects and/or toxicities is screening for,monitoring, reducing, preventing, attenuating, and/or mitigating bonedensity loss and/or fracture risk. Often bone density loss isasymptomatic and/or early signs of skeletal-related side effects are notevident with, for example, bone density scanning.

Bone metabolism is a continuous dual process of bone formation and bonedestruction. Bone destruction is referred to as bone resorption and iscarried out by osteoclasts, while bone formation is carried out byosteoblasts. In adults, the dual processes of bone formation and bonedestruction are in balance, maintaining a constant, homeostaticallycontrolled amount of bone. Bone metabolism may be assessed and/ormonitored by measurement of biomarkers (e.g., enzymes, proteins, and/ordegradation products) released during bone formation and boneresorption. These biomarkers are often referred to as “bone turnovermarkers”, and include bone formation markers and bone resorptionmarkers. Bone formation biomarkers include serum total alkalinephosphatase, serum bone-specific alkaline phosphatase, serumosteocalcin, serum procollagen type 1 amino-terminal propeptide (P1NP)and serum procollagen type 1 carboxy-terminal propeptide (P1CP). Boneresorption biomarkers include, urinary hydroxyproline, urinary totalpyridinoline (PYD), urinary free deoxypryidinoline (DPD), urinarycollagen type 1 cross-linked N-telopeptide (NTX), urinary or serumcollagen type 1 cross-linked C-telopeptide (CTX), bone sialoprotein(BSP), and tartrate-resistant acid phosphatase 5b.

Approximately 90% of the organic matrix of bone is type 1 collagen, ahelical protein that is cross-linked at the N- and C-terminal ends ofthe molecule. During bone resorption, osteoclasts secrete a mixture ofacid and neutral proteases that degrade the collagen fibrils intomolecular fragments including C-telopeptide (CTX). As bone ages, thealpha form of aspartic acid present in CTX converts to the beta form(β-CTX). β-CTX is released into the bloodstream during bone resorptionand serves as a specific marker for the degradation of mature type 1collagen.

Bone turnover markers have been used to monitor anti-resorptivetherapies (e.g., hormone replacement therapies and bisphosphonatetherapies) in post-menopausal women, as well as in individuals diagnosedwith osteopenia. In addition, bone turnover markers may be used toassess drug-induced osteoporosis resulting from therapy with hormonaland non-hormonal drugs. These drugs may include, but are not limited to,glucocorticoids, thyroid hormone, aromatase inhibitors, ovariansuppressing agents, androgen deprivation therapy, thiazolidinediones,selective serotonin reuptake inhibitors, anticonvulsants, heparins, oralanticoagulants, loop diuretics, calcineurin inhibitors, anti-retroviraltherapy, and proton pump inhibitors. Bone turnover markers have notpreviously been used to assess the effect of Wnt pathway inhibitors.Accordingly, in some embodiments, the present invention provides methodsfor using bone turnover markers to monitor skeletal-related side effectsand/or toxicities in subjects being treated with a Wnt pathwayinhibitor. In some embodiments, the methods use a bone formationbiomarker to monitor and/or detect decreased levels of bone formation.In some embodiments, the methods use a bone resorption biomarker tomonitor and/or detect increased levels of bone resorption. In someembodiments, monitoring the level of a bone formation biomarker gives anearly indication of decreased levels of bone formation and/or increasedrisk of bone fracture, osteopenia, and/or osteoporosis. In someembodiments, monitoring the level of a bone resorption biomarker givesan early indication of increased levels of bone resorption and/orincreased risk of bone fracture, osteopenia, and/or osteoporosis. Insome embodiments, the methods detect skeletal-related side effectsand/or toxicities prior to any evidence of skeletal dysfunction asevaluated by bone density scans.

In certain embodiments, the skeletal-related side effects and/ortoxicities that are detected, identified, monitored, reduced, prevented,attenuated, and/or screened for are skeletal-related side effects and/ortoxicities caused by, associated with, and/or related to administrationof a Wnt pathway inhibitor or treatment with a Wnt pathway inhibitor. Incertain embodiments, the skeletal-related side effects and/or toxicitiesare related to the Wnt pathway inhibitor. In certain embodiments, theskeletal-related side effects and/or toxicities are related to theactivity of the Wnt pathway inhibitor. In certain embodiments, theskeletal-related side effects and/or toxicities are related to a Wntpathway inhibitor that is an anti-FZD antibody. In certain embodiments,the skeletal-related side effects and/or toxicities are related to a Wntpathway inhibitor that is anti-FZD antibody OMP-18R5. In certainembodiments, the skeletal-related side effects and/or toxicities arerelated to the Wnt pathway inhibitor that is a FZD soluble receptor. Incertain embodiments, the skeletal-related side effects and/or toxicitiesare related to the Wnt pathway inhibitor that is a FZD8-Fc solublereceptor. In certain embodiments, the skeletal-related side effectsand/or toxicities are related to the Wnt pathway inhibitor that isFZD8-Fc soluble receptor 54F28.

The invention provides methods for selecting a subject for treatmentwith a Wnt pathway inhibitor, comprising: determining the level of abiomarker in a sample, and selecting the subject for treatment with theWnt pathway inhibitor if the level of the biomarker is below apredetermined level. In some embodiments, the methods for selecting asubject for treatment with a Wnt pathway inhibitor comprise: obtaining abiological sample from the subject, determining the level of a biomarkerin the sample, and selecting the subject for treatment with the Wntpathway inhibitor if the level of the biomarker is below a predeterminedlevel. In some embodiments, the biomarker is a bone turnover marker. Insome embodiments, the bone turnover marker is a bone resorptionbiomarker. In some embodiments, the bone resorption biomarker is β-CTX.

In some embodiments, the method of selecting a subject for treatmentwith a Wnt pathway inhibitor comprises: obtaining a biological samplefrom the subject, determining the level of a bone turnover marker in thesample, and selecting the subject for treatment with the Wnt pathwayinhibitor if the level of the bone turnover marker is below apredetermined level. In some embodiments, the biological sample isurine, blood, serum, or plasma. In some embodiments, the bone turnovermarker is a bone resorptive biomarker. In some embodiments, the boneresorption biomarker is urinary hydroxyproline, urinary totalpyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinarycollagen type 1 cross-linked N-telopeptide (NTX), urinary or serumcollagen type 1 cross-linked C-telopeptide (CTX), bone sialoprotein(BSP), or tartrate-resistant acid phosphatase 5b. In some embodiments,the bone resorptive biomarker is CTX or β-CTX. Thus, in someembodiments, the methods of selecting a subject for treatment with a Wntpathway inhibitor, comprising: obtaining a biological sample from thesubject, determining the level of β-CTX in the sample, and selecting thesubject for treatment with the Wnt pathway inhibitor if the level ofβ-CTX is below a predetermined level.

The invention provides methods of identifying a subject as eligible fortreatment with a Wnt pathway inhibitor, comprising: determining thelevel of a biomarker in a sample, and identifying the subject aseligible for treatment with the Wnt pathway inhibitor if the level ofthe biomarker is below a predetermined level. In some embodiments, themethods of identifying a subject as eligible for treatment with a Wntpathway inhibitor comprise: obtaining a biological sample from thesubject, determining the level of a biomarker in the sample, andidentifying the subject as eligible for treatment with the Wnt pathwayinhibitor if the level of the biomarker is below a predetermined level.In some embodiments, the biomarker is a bone turnover marker. In someembodiments, the biomarker is a bone resorption biomarker. In someembodiments, the bone resorption biomarker is urinary hydroxyproline,urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD),urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary orserum collagen type 1 cross-linked C-telopeptide (CTX), bonesialoprotein (BSP), or tartrate-resistant acid phosphatase 5b. In someembodiments, the bone resorption biomarker is CTX. In some embodiments,the bone resorption biomarker is β-CTX. In some embodiments, the methodsof identifying a subject as eligible for treatment with a Wnt pathwayinhibitor comprise: obtaining a biological sample from the subject,determining the level of β-CTX in the sample, and identifying thesubject as eligible for treatment with the Wnt pathway inhibitor if thelevel of β-CTX is below a predetermined level.

The invention also provides methods of monitoring a subject receivingtreatment with a Wnt pathway inhibitor for the development ofskeletal-related side effects and/or toxicity, comprising: determiningthe level of a biomarker in a sample, and comparing the level of thebiomarker in the sample to a predetermined level of the biomarker,wherein an increase in the level of the biomarker indicates developmentof skeletal-related side effects and/or toxicity. In some embodiments,the methods of monitoring a subject receiving treatment with a Wntpathway inhibitor for the development of skeletal-related side effectsand/or toxicity comprise: obtaining a biological sample from the subjectreceiving treatment, determining the level of a biomarker in the sample,and comparing the level of the biomarker in the sample to apredetermined level of the biomarker, wherein an increase in the levelof the biomarker indicates development of skeletal-related side effectsand/or toxicity. In some embodiments, the skeletal-related side effectand/or toxicity is an increased risk of bone fracture. In someembodiments, the skeletal-related side effect and/or toxicity isosteopenia or osteoporosis. In some embodiments, the biomarker is a boneturnover marker. In some embodiments, the biomarker is a bone resorptionbiomarker. In some embodiments, the bone resorption biomarker is urinaryhydroxyproline, urinary total pyridinoline (PYD), urinary freedeoxypyridinoline (DPD), urinary collagen type 1 cross-linkedN-telopeptide (NTX), urinary or serum collagen type 1 cross-linkedC-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acidphosphatase 5b. In some embodiments, the bone resorption biomarker isCTX. In some embodiments, the bone resorption biomarker is β-CTX. Insome embodiments, a method of monitoring a subject receiving treatmentwith a Wnt pathway inhibitor for the development of skeletal-relatedside effects and/or toxicity, comprises: obtaining a biological samplefrom the subject receiving treatment, determining the level of β-CTX inthe sample, and comparing the level of β-CTX in the sample to apredetermined level of β-CTX, wherein an increase in the level of β-CTXindicates development of skeletal-related side effects and/or toxicity.

The invention also provides methods of detecting the development ofskeletal-related side effects and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor, comprising: determining thelevel of a biomarker in a sample, and comparing the level of a biomarkerin the sample to a predetermined level of the biomarker, wherein anincrease in the level of the biomarker indicates development ofskeletal-related side effects and/or toxicity. In some embodiments, themethods of detecting the development of skeletal-related side effectsand/or toxicity in a subject receiving treatment with a Wnt pathwayinhibitor comprise: obtaining a biological sample from the subjectreceiving treatment, determining the level of a biomarker in the sample,and comparing the level of a biomarker in the sample to a predeterminedlevel of the biomarker, wherein an increase in the level of thebiomarker indicates development of skeletal-related side effects and/ortoxicity. In some embodiments, the skeletal-related side effect and/ortoxicity is an increased risk of bone fracture. In some embodiments, theskeletal-related side effect and/or toxicity is osteopenia orosteoporosis. In some embodiments, the biomarker is a bone turnovermarker. In some embodiments, the biomarker is a bone resorptionbiomarker. In some embodiments, the bone resorption biomarker is urinaryhydroxyproline, urinary total pyridinoline (PYD), urinary freedeoxypyridinoline (DPD), urinary collagen type 1 cross-linkedN-telopeptide (NTX), urinary or serum collagen type 1 cross-linkedC-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acidphosphatase 5b. In some embodiments, the bone resorption biomarker isCTX. In some embodiments, the bone resorption biomarker is β-CTX. Insome embodiments, the methods of detecting the development ofskeletal-related side effects and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor comprise: obtaining a biologicalsample from the subject receiving treatment, determining the level ofβ-CTX in the sample, and comparing the level of β-CTX in the sample to apredetermined level of β-CTX, wherein an increase in the level of β-CTXindicates development of skeletal-related side effects and/or toxicity.

The invention also provides methods for identifying skeletal-relatedside effects and/or toxicity in a subject receiving treatment with a Wntpathway inhibitor, comprising: determining the level of a biomarker in asample, and comparing the level of the biomarker in the sample to apredetermined level of the biomarker, wherein if the level of thebiomarker in the sample is higher than the predetermined level of thebiomarker then a skeletal-related side effect and/or toxicity isindicated. In some embodiments, the methods for identifyingskeletal-related side effects and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor comprise: obtaining a biologicalsample from the subject receiving treatment, determining the level of abiomarker in the sample, and comparing the level of the biomarker in thesample to a predetermined level of the biomarker, wherein if the levelof the biomarker in the sample is higher than the predetermined level ofthe biomarker then a skeletal-related side effect and/or toxicity isindicated. In some embodiments, the skeletal-related side effect and/ortoxicity is an increased risk of bone fracture. In some embodiments, theskeletal-related side effect and/or toxicity is osteopenia orosteoporosis. In some embodiments, the biomarker is a bone turnovermarker. In some embodiments, the biomarker is a bone resorptionbiomarker. In some embodiments, the bone resorption biomarker is urinaryhydroxyproline, urinary total pyridinoline (PYD), urinary freedeoxypyridinoline (DPD), urinary collagen type 1 cross-linkedN-telopeptide (NTX), urinary or serum collagen type 1 cross-linkedC-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acidphosphatase 5b. In some embodiments, the bone resorption biomarker isCTX. In some embodiments, the bone resorption biomarker is β-CTX. Insome embodiments, a method for identifying a skeletal-related sideeffect and/or toxicity in a subject receiving treatment with a Wntpathway inhibitor comprises: obtaining a biological sample from thesubject receiving treatment, determining the level of β-CTX in thesample, and comparing the level of β-CTX in the sample to apredetermined level of β-CTX, wherein if the level of β-CTX in thesample is higher than the predetermined level of β-CTX then askeletal-related side effect and/or toxicity is indicated.

The invention also provides methods for monitoring skeletal-related sideeffects and/or toxicity in a subject receiving treatment with a Wntpathway inhibitor, comprising: determining the level of a biomarker in asample, and comparing the level of the biomarker in the sample to apredetermined level of the biomarker, wherein if the level of thebiomarker in the sample is higher than the predetermined level of thebiomarker then skeletal-related side effects and/or toxicity isindicated. In some embodiments, the methods for monitoringskeletal-related side effects and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor comprise: obtaining a biologicalsample from the subject receiving treatment, determining the level of abiomarker in the sample, and comparing the level of the biomarker in thesample to a predetermined level of the biomarker, wherein if the levelof the biomarker in the sample is higher than the predetermined level ofthe biomarker then skeletal-related side effects and/or toxicity isindicated. In some embodiments, the skeletal-related side effect and/ortoxicity is an increased risk of bone fracture. In some embodiments, theskeletal-related side effect and/or toxicity is osteopenia orosteoporosis. In some embodiments, the biomarker is a bone turnovermarker. In some embodiments, the biomarker is a bone resorptionbiomarker. In some embodiments, the bone resorption biomarker is urinaryhydroxyproline, urinary total pyridinoline (PYD), urinary freedeoxypyridinoline (DPD), urinary collagen type 1 cross-linkedN-telopeptide (NTX), urinary or serum collagen type 1 cross-linkedC-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acidphosphatase 5b. In some embodiments, the bone resorption biomarker isCTX. In some embodiments, the bone resorption biomarker is β-CTX. Insome embodiments, a method for monitoring cardiotoxicity in a subjectreceiving treatment with a Wnt pathway inhibitor comprises: obtaining abiological sample from the subject receiving treatment, determining thelevel of β-CTX in the sample, and comparing the level of β-CTX in thesample to a predetermined level of β-CTX, wherein if the level of β-CTXin the sample is higher than the predetermined level of β-CTX then askeletal-related side effect and/or toxicity is indicated.

The invention also provides methods of reducing skeletal-related sideeffects and/or toxicity in a subject receiving treatment with a Wntpathway inhibitor, comprising: determining the level of a biomarker in asample from the subject, comparing the level of the biomarker in thesample to a predetermined level of the biomarker, and administering tothe subject a therapeutically effective amount of an anti-resorptivemedication such as a bisphosphonate if the level of the biomarker in thesample is higher than the predetermined level of the biomarker. In someembodiments, the methods of reducing skeletal-related side effectsand/or toxicity in a subject receiving treatment with a Wnt pathwayinhibitor comprise: obtaining a biological sample from the subjectreceiving treatment, determining the level of a biomarker in the sample,comparing the level of the biomarker in the sample to a predeterminedlevel of the biomarker, and administering to the subject atherapeutically effective amount of an anti-resorptive medication suchas a bisphosphonate if the level of the biomarker in the sample ishigher than the predetermined level of the biomarker. In someembodiments, the skeletal-related side effect and/or toxicity is anincreased risk of bone fracture. In some embodiments, theskeletal-related side effect and/or toxicity is osteopenia orosteoporosis. In some embodiments, the biomarker is a bone turnovermarker. In some embodiments, the biomarker is a bone resorptionbiomarker. In some embodiments, the bone resorption biomarker is urinaryhydroxyproline, urinary total pyridinoline (PYD), urinary freedeoxypyridinoline (DPD), urinary collagen type 1 cross-linkedN-telopeptide (NTX), urinary or serum collagen type 1 cross-linkedC-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acidphosphatase 5b. In some embodiments, the bone resorption biomarker isCTX. In some embodiments, the bone resorption biomarker is β-CTX. Insome embodiments, a method for reducing skeletal-related side effectsand/or toxicity in a subject receiving treatment with a Wnt pathwayinhibitor comprises: obtaining a biological sample from the subjectreceiving treatment, determining the level of β-CTX in the sample, andcomparing the level of β-CTX in the sample to a predetermined level ofβ-CTX, and administering to the subject a therapeutically effectiveamount of an anti-resorptive medication if the level of β-CTX in thesample is higher than the predetermined level of β-CTX. In someembodiments, the anti-resorptive medication is a bisphosphonate.

The invention also provides methods of preventing or attenuating thedevelopment of skeletal-related side effects and/or toxicity in asubject receiving treatment with a Wnt pathway inhibitor, comprising:determining the level of a biomarker in a sample from the subject,comparing the level of the biomarker in the sample to a predeterminedlevel of the biomarker; administering to the subject a therapeuticallyeffective amount of an anti-resorptive medication, and administering tothe subject the Wnt pathway inhibitor. In some embodiments, the methodsof preventing or attenuating the development of skeletal-related sideeffects and/or toxicity in a subject receiving treatment with a Wntpathway inhibitor comprise: obtaining a biological sample from thesubject prior to treatment with the Wnt pathway inhibitor, determiningthe level of a biomarker in the sample, comparing the level of thebiomarker in the sample to a predetermined level of the biomarker;administering to the subject a therapeutically effective amount of ananti-resorptive medication, and administering to the subject the Wntpathway inhibitor. In some embodiments, the skeletal-related side effectand/or toxicity is an increased risk of bone fracture. In someembodiments, the skeletal-related side effect and/or toxicity isosteopenia or osteoporosis. In some embodiments, the biomarker is a boneturnover marker. In some embodiments, the biomarker is a bone resorptionbiomarker. In some embodiments, the bone resorption biomarker is urinaryhydroxyproline, urinary total pyridinoline (PYD), urinary freedeoxypyridinoline (DPD), urinary collagen type 1 cross-linkedN-telopeptide (NTX), urinary or serum collagen type 1 cross-linkedC-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acidphosphatase 5b. In some embodiments, the bone resorption biomarker isCTX. In some embodiments, the bone resorption biomarker is β-CTX. Insome embodiments, a method of preventing or attenuating the developmentof a skeletal-related side effect and/or toxicity in a subject receivingtreatment with a Wnt pathway inhibitor comprises: obtaining a biologicalsample from the subject prior to treatment with the Wnt pathwayinhibitor, determining the level of β-CTX in the sample, comparing thelevel of β-CTX in the sample to a predetermined level of β-CTX;administering to the subject a therapeutically effective amount of ananti-resorptive medication if the level of β-CTX in the sample is higherthan the predetermined level of β-CTX; and administering to the subjectthe Wnt pathway inhibitor.

In some embodiments of the methods described herein, the predeterminedlevel is about 1500 pg/ml or less in a blood, serum, or plasma sample.In some embodiments, the predetermined level is about 1200 pg/ml or lessin a blood, serum, or plasma sample. In some embodiments, thepredetermined level is about 1000 pg/ml or less in a blood, serum, orplasma sample. In some embodiments, the predetermined level is about 800pg/ml or less in a blood, serum, or plasma sample. In some embodiments,the predetermined level is about 600 pg/ml or less in a blood, serum, orplasma sample. In some embodiments, the predetermined level is about 400pg/ml or less in a blood, serum, or plasma sample. In the context ofpredetermined levels of β-CTX, the term “about” means the referencedamount plus or minus 10% of that referenced amount.

In some embodiments, the predetermined level of a biomarker (e.g., abone resorption biomarker or β-CTX) is the amount of the biomarker in asample obtained at an earlier date. In some embodiments, thepredetermined level of a biomarker (e.g., a bone resorption biomarker orβ-CTX) is the amount of the biomarker in a sample obtained at an initialscreening. In some embodiments, the predetermined level of a biomarker(e.g., a bone resorption biomarker or β-CTX) is the amount of thebiomarker in a sample obtained prior to treatment. In some embodiments,the predetermined level of a biomarker (e.g., a bone resorptionbiomarker or β-CTX) is the amount of the biomarker in a sample obtainedat an initial screening. In some embodiments, the predetermined level ofa biomarker (e.g., a bone resorption biomarker or β-CTX) is a normalreference level. In some embodiments, the predetermined level of abiomarker (e.g., a bone resorption biomarker or β-CTX) is a baselinelevel. In some embodiments, the baseline level is the amount of thebiomarker determined at an initial screening. In some embodiments, thebaseline level is the amount of the biomarker determined prior totreatment.

In some embodiments, if the β-CTX level in the sample is increased2-fold or greater (i.e., a doubling or greater) as compared to apredetermined level, the subject is administered a therapeuticallyeffective amount of an anti-resorptive medication. In some embodiments,if the β-CTX level in the sample is increased 2-fold or greater (i.e., adoubling or greater) as compared to a baseline level, the subject isadministered a therapeutically effective amount of an anti-resorptivemedication.

In any of the methods described herein, a biological sample is obtainedapproximately every week, every 2 weeks, every 3 weeks, every 4 weeks,every 5 weeks, or every 6 weeks.

In some embodiments of any of the methods described herein, the subjectsare evaluated using a DEXA (dual energy X-ray absorptiometry) bonedensity scan. This technique is the most commonly used test formeasuring bone mineral density (BMD). The DEXA output includes aT-score, which compares the subject's bone density to a 30-35 year oldperson, and a Z-score, which compares the subject's bone density to theaverage bone density of someone their age and gender. The T-score isused to determine if an individual has osteopenia or osteoporosisaccording to a standard scale. A T-score greater than −1 is considerednormal bone density; a T-score between −1 and −2.5, is consideredosteopenia; a T-score less than −2.5 is considered osteoporosis; and aT-score less than −2.5 and 1+ osteoporotic fractures is consideredsevere (established) osteoporosis. In some embodiments, askeletal-related side effect and/or toxicity is indicated if the T-scoredeclines to less than −2.5 in the total femur or vertebrae L1-L4. Insome embodiments, a skeletal-related side effect and/or toxicity isindicated if the T-score declines to less than −2.0 in the total femuror vertebrae L1-L4. In some embodiments, a skeletal-related side effectand/or toxicity is indicated if the T-score declines to less than −1.5in the total femur or vertebrae L1-L4. In some embodiments, askeletal-related side effect and/or toxicity is indicated if the T-scoredeclines to less than −1.0 in the total femur or vertebrae L1-L4.

The invention also provides methods of ameliorating skeletal-relatedside effects and/or toxicity in a subject administered a Wnt pathwayinhibitor, comprising: administering to the subject a therapeuticallyeffective amount of an anti-resorptive medication.

The invention also provides methods of screening a subject for the riskof skeletal-related side effects and/or toxicity from treatment with aWnt pathway inhibitor, comprising: determining the level of a biomarkerin a sample from the subject, and comparing the level of the biomarkerin the sample to a predetermined level of the biomarker, wherein if thelevel of the biomarker in the sample is higher than the predeterminedlevel of the biomarker then the subject is at risk for skeletal-relatedside effects and/or toxicity. In some embodiments, the methods ofscreening a subject for the risk of skeletal-related side effects and/ortoxicity from treatment with a Wnt pathway inhibitor comprise: obtaininga biological sample from the subject prior to treatment with the Wntpathway inhibitor, determining the level of a biomarker in the sample,and comparing the level of the biomarker in the sample to apredetermined level of the biomarker, wherein if the level of thebiomarker in the sample is higher than the predetermined level of thebiomarker then the subject is at risk for skeletal-related side effectsand/or toxicity. In some embodiments, the skeletal-related side effectand/or toxicity is an increased risk of bone fracture. In someembodiments, the skeletal-related side effect and/or toxicity isosteopenia or osteoporosis. In some embodiments, the biomarker is a boneturnover marker. In some embodiments, the biomarker is a bone resorptionbiomarker. In some embodiments, the bone resorption biomarker is urinaryhydroxyproline, urinary total pyridinoline (PYD), urinary freedeoxypyridinoline (DPD), urinary collagen type 1 cross-linkedN-telopeptide (NTX), urinary or serum collagen type 1 cross-linkedC-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acidphosphatase 5b. In some embodiments, the bone resorption biomarker isCTX. In some embodiments, the bone resorption biomarker is β-CTX. Insome embodiments, a method of screening a subject for the risk of askeletal-related side effect and/or toxicity from treatment with a Wntpathway inhibitor comprises: obtaining a biological sample from thesubject prior to treatment with the Wnt pathway inhibitor, determiningthe level of β-CTX in the sample, and comparing the level of β-CTX inthe sample to a predetermined level of β-CTX, wherein if the level ofβ-CTX in the sample is higher than the predetermined level of β-CTX thenthe subject is at risk for a skeletal-related side effect and/ortoxicity. In some embodiments, the predetermined level of β-CTX is avalue determined at an initial screening. In some embodiments, thepredetermined level of β-CTX is from about 400 to 1200 pg/ml. In someembodiments, if the subject is at risk for a skeletal-related sideeffect and/or toxicity, the subject is administered a therapeuticallyeffective amount of an anti-resorptive medication prior to treatmentwith the Wnt pathway inhibitor.

In some embodiments of the methods described herein, the anti-resorptivemedication is a bisphosphonate. It is believed that bisphosphonatesprevent loss of bone mass by “inducing” osteoclasts to undergo apoptosisand thereby inhibiting the digestion of bone. In some embodiments, thebisphosphonate is selected from the group consisting of: etidronate,clodronate, tiludronate, pamidronate, neridronate, olpadronate,alendronate (FOSAMAX), ibandronate (BONIVA), risedronate (ACTONEL), andzoledronic acid (RECLAST). In some embodiments, the bisphosphonate iszoledronic acid. In some embodiments, the anti-resorptive medication isanti-RANKL antibody denosumab (PROLIA).

In any of the methods described herein, the Wnt pathway inhibitor is ananti-FZD antibody. In any of the methods described herein, the Wntpathway inhibitor is an anti-Wnt antibody. In any of the methodsdescribed herein, the Wnt pathway inhibitor is a FZD soluble receptor.

In certain embodiments of any of the methods described herein, the Wntpathway inhibitor is an antibody comprising: (a) a heavy chain CDR1comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2 comprisingVISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprisingNFIKYVFAN (SEQ ID NO:3), and (b) a light chain CDR1 comprisingSGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprising DKSNRPSG (SEQID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:6).

In certain embodiments of any of the methods described herein, the Wntpathway inhibitor is an antibody comprising a heavy chain variableregion comprising SEQ ID NO:7 and a light chain variable regioncomprising SEQ ID NO:8.

In certain embodiments, the Wnt pathway inhibitor comprises the sameheavy chain variable region and the same light chain variable regionsequences as OMP-18R5. In some embodiments, the Wnt pathway inhibitor isantibody OMP-18R5. OMP-18R5 is an IgG2 human monoclonal antibody thatbinds human FZD1, FZD2, FZD5, FZD7, and FZD8 receptors and has beenpreviously described in U.S. Pat. No. 7,982,013.

In certain embodiments, the Wnt pathway inhibitor comprises the sameheavy and light chain amino acid sequences as an antibody encoded by aplasmid deposited with ATCC having deposit no. PTA-9541. In certainembodiments, the Wnt pathway inhibitor is encoded by the plasmid havingATCC deposit no. PTA-9541 which was deposited with American Type CultureCollection (ATCC), at 10801 University Boulevard, Manassas, Va., 20110,under the conditions of the Budapest Treaty on Sep. 29, 2008. In certainembodiments, the Wnt pathway inhibitor competes for specific binding toa human FZD with an antibody encoded by the plasmid deposited with ATCChaving deposit no. PTA-9541.

In certain embodiments of any of the methods described herein, the Wntpathway inhibitor is a FZD soluble receptor. In some embodiments, theWnt pathway inhibitor is a FZD8 soluble receptor comprising SEQ IDNO:20, SEQ ID NO:30, or SEQ ID NO:33. In some embodiments, the Wntpathway inhibitor is a FZD8 soluble receptor comprising SEQ ID NO:20. Insome embodiments, the Wnt pathway inhibitor is a FZD8 soluble receptorcomprising SEQ ID NO:30. In some embodiments, the Wnt pathway inhibitoris a FZD8 soluble receptor comprising SEQ ID NO:33.

In certain embodiments of any of the methods described herein, the Wntpathway inhibitor is a FZD-Fc soluble receptor. In some embodiments, theWnt pathway inhibitor is a FZD8-Fc soluble receptor. In someembodiments, the Wnt pathway inhibitor is a FZD8-Fc soluble receptorcomprising SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:41. In someembodiments, the Wnt pathway inhibitor is a FZD8-Fc soluble receptorcomprising SEQ ID NO:39. In some embodiments, the Wnt pathway inhibitoris a FZD8-Fc soluble receptor comprising SEQ ID NO:40. In someembodiments, the Wnt pathway inhibitor is a FZD8-Fc soluble receptorcomprising SEQ ID NO:41. In some embodiments, the Wnt pathway inhibitoris OMP-54F28. In some embodiments, the Wnt pathway inhibitor is notOMP-54F28.

In some embodiments, the subject has cancer. In some embodiments, thecancer is selected from the group consisting of: lung cancer, breastcancer, colon cancer, colorectal cancer, melanoma, pancreatic cancer,gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer,endometrial cancer, kidney cancer, prostate cancer, thyroid cancer,neuroendocrine cancer, neuroblastoma, glioma, glioblastoma multiforme,cervical cancer, stomach cancer, bladder cancer, hepatoma, and head andneck cancer. As used herein, “lung cancer” refers to non-small cell lungcancer (NSCLC) and small cell lung cancer (SCLC). In certainembodiments, the cancer is a hematological cancer, such as a lymphoma orleukemia. In certain embodiments, the cancer is NSCLC. In certainembodiments, the cancer is ovarian cancer. In certain embodiments, thecancer is pancreatic cancer. In certain embodiments, the cancer is not aneuroendocrine cancer.

Thus, the invention also provides methods of treating cancer. In someembodiments, the methods comprise a method of treating cancer in asubject in need thereof, comprising: (a) administering to the subject atherapeutically effective amount of a Wnt pathway inhibitor; and (b)determining the level of a bone resorption biomarker in a sample fromthe subject. In some embodiments, a method of treating cancer comprises(a) administering to the subject a therapeutically effective amount of aWnt pathway inhibitor; (b) determining the level of a bone resorptionbiomarker in a sample from the subject; and (c) comparing the level ofthe bone resorption biomarker in the sample to a predetermined level ofthe bone resorption biomarker. In some embodiments, a method of treatingcancer comprises (a) administering to the subject a therapeuticallyeffective amount of a Wnt pathway inhibitor; (b) determining the levelof a bone resorption biomarker in a sample from the subject; and (c)comparing the level of the bone resorption biomarker in the sample to apredetermined level of the bone resorption biomarker; wherein if thelevel of the bone resorption biomarker in the sample is higher than thepredetermined level of the bone resorption biomarker then the subject isat risk for a skeletal-related side effect and/or toxicity. In someembodiments, a method of treating cancer comprises (a) administering tothe subject a therapeutically effective amount of a Wnt pathwayinhibitor; (b) determining the level of a bone resorption biomarker in asample from the subject; and (c) comparing the level of the boneresorption biomarker in the sample to a predetermined level of the boneresorption biomarker; wherein if the level of the bone resorptionbiomarker in the sample is higher than the predetermined level of thebone resorption biomarker then the subject is administered atherapeutically effective amount of an anti-resorptive medication.

The invention also provides methods of inhibiting tumor growth. In someembodiments, the methods comprise a method of inhibiting tumor growth ina subject in need thereof, comprising: (a) administering to the subjecta therapeutically effective amount of a Wnt pathway inhibitor; and (b)determining the level of a bone resorption biomarker in a sample fromthe subject. In some embodiments, a method of inhibiting tumor growthcomprises (a) administering to the subject a therapeutically effectiveamount of a Wnt pathway inhibitor; (b) determining the level of a boneresorption biomarker in a sample from the subject; and (c) comparing thelevel of the bone resorption biomarker in the sample to a predeterminedlevel of the bone resorption biomarker. In some embodiments, a method ofinhibiting tumor growth comprises (a) administering to the subject atherapeutically effective amount of a Wnt pathway inhibitor; (b)determining the level of a bone resorption biomarker in a sample fromthe subject; and (c) comparing the level of the bone resorptionbiomarker in the sample to a predetermined level of the bone resorptionbiomarker; wherein if the level of the bone resorption biomarker in thesample is higher than the predetermined level of the bone resorptionbiomarker then the subject is at risk for a skeletal-related side effectand/or toxicity. In some embodiments, a method of inhibiting tumorgrowth comprises (a) administering to the subject a therapeuticallyeffective amount of a Wnt pathway inhibitor; (b) determining the levelof a bone resorption biomarker in a sample from the subject; and (c)comparing the level of the bone resorption biomarker in the sample to apredetermined level of the bone resorption biomarker; wherein if thelevel of the bone resorption biomarker in the sample is higher than thepredetermined level of the bone resorption biomarker then the subject isadministered a therapeutically effective amount of an anti-resorptivemedication.

In some embodiments, the biological sample is a body fluid. In someembodiments, the biological sample is blood, plasma, serum, or urine. Insome embodiments, the biological sample is a venous whole bloodspecimen. In some embodiments, the biological sample is a venous wholeblood specimen using EDTA or heparin as an anticoagulant. In someembodiments, the biological sample is a plasma specimen. In someembodiments, the biological sample is a plasma specimen using EDTA orheparin as an anticoagulant. Samples of body fluids may be obtained byany method known in the art. In some embodiments, the biological sampleis a frozen tissue sample or is fresh tissue sample.

Assays for measuring or determining the level of a bone resorptionbiomarker (e.g., β-CTX) in a sample are known to those of skilled in theart. For example, in some embodiments an immunoassay that quantitativelymeasures β-CTX levels in whole blood or plasma specimens is used. Insome embodiments, the sample contains EDTA as an anticoagulant. In someembodiments, the sample contains heparin as an anticoagulant. In someembodiments, the immunoassay comprises two highly specific monoclonalantibodies against the amino acid sequence of EKAHD-β-GGR of β-CTX,wherein the aspartic acid residue is β-isomerized. In order to obtain aspecific signal in the immunoassay, two chains of EKAHD-β-GGR must becross-linked. In some embodiments, a sample and appropriate controls areplaced into streptavidin-coated microtiter wells, followed by a solutioncontaining biotinylated monoclonal antibodies against the amino acidsequence of EKAHD-β-GGR of β-CTX. After incubation and washing, achromogenic substrate solution is added to microtiter wells. Afterincubation, the reaction is stopped. Absorbance of the microtiter wellsis read and the β-CTX concentration is determined.

In some embodiments, the Wnt pathway inhibitor is administered as aninitial dose of about 0.5 mg/kg. For example, antibody OMP-18R5 isdiluted with 5% dextrose in water (USP) to a total volume of 250 mL. TheOMP-18R5 is delivered through a 0.22-micron filter over 30 minutes as anintravenous infusion. In some embodiments, subsequent doses areadministered in a similar manner.

In another aspect of the invention, the methods described herein mayfurther comprise administering one or more additional therapeuticagents. An additional therapeutic agent can be administered prior to,concurrently with, and/or subsequently to, administration of the Wntpathway inhibitor. Pharmaceutical compositions comprising a Wnt pathwayinhibitor and an additional therapeutic agent(s) are also provided. Insome embodiments, the one or more additional therapeutic agents comprise1, 2, 3, or more additional therapeutic agents.

Combination therapy with at least two therapeutic agents often usesagents that work by different mechanisms of action, although this is notrequired. Combination therapy using agents with different mechanisms ofaction may result in additive or synergetic effects. Combination therapymay allow for a lower dose of each agent than is used in monotherapy,thereby reducing side effects and/or toxicities. Combination therapy mayincrease the therapeutic index of one or both of the therapeutic agents.Combination therapy may decrease the likelihood that resistant cancercells will develop. In some embodiments, combination therapy comprises atherapeutic agent that primarily affects (e.g., inhibits or kills)non-tumorigenic cells and a therapeutic agent that primarily affects(e.g., inhibits or kills) tumorigenic CSCs.

Therapeutic agents that may be administered in combination with the Wntpathway inhibitor include chemotherapeutic agents. Thus, in someembodiments, the method or treatment involves the administration of aWnt pathway inhibitor of the present invention in combination with achemotherapeutic agent or cocktail of multiple differentchemotherapeutic agents. Treatment with a Wnt pathway inhibitor (e.g.,an antibody or soluble receptor) can occur prior to, concurrently with,or subsequent to administration of chemotherapies. Combinedadministration can include co-administration, either in a singlepharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously. Preparation and dosing schedules for suchchemotherapeutic agents can be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in The Chemotherapy Source Book, 4th Edition, 2008, M. C.Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

Chemotherapeutic agents useful in the instant invention include, but arenot limited to, alkylating agents such as thiotepa and cyclophosphamide(CYTOXAN); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenishers suchas folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel(TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine (XELODA); and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including, for example, tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above. In certain embodiments, theadditional therapeutic agent is cisplatin. In certain embodiments, theadditional therapeutic agent is carboplatin. In certain embodiments, theadditional therapeutic agent is paclitaxel. In certain embodiments,where the chemotherapeutic agent administered in combination with a Wntpathway inhibitor is carboplatin, the cancer or tumor being treated islung cancer or a lung tumor.

In certain embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapeutic agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D,etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese. In certain embodiments, the additional therapeutic agent isirinotecan.

In certain embodiments, the chemotherapeutic agent is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside,thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine,6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, aswell as pharmaceutically acceptable salts, acids, or derivatives of anyof these. In certain embodiments, the additional therapeutic agent isgemcitabine. In some embodiments, the additional therapeutic agent ispemetrexed. In certain embodiments, where the chemotherapeutic agentadministered in combination with a Wnt pathway inhibitor is gemcitabine,the cancer or tumor being treated is pancreatic cancer or a pancreatictumor. In certain embodiments, where the chemotherapeutic agentadministered in combination with a Wnt pathway inhibitor is pemetrexed,the cancer or tumor being treated is lung cancer or a lung tumor. Insome embodiments, the Wnt pathway inhibitor is administered incombination with pemetrexed and carboplatin.

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin. In someembodiments, the agent is a taxane. In certain embodiments, the agent ispaclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, orderivative of paclitaxel or docetaxel. In certain embodiments, the agentis paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel(ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternativeembodiments, the antimitotic agent comprises a vinca alkaloid, such asvincristine, binblastine, vinorelbine, or vindesine, or pharmaceuticallyacceptable salts, acids, or derivatives thereof. In some embodiments,the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor ofa mitotic kinase such as Aurora A or Plk1. In certain embodiments, wherethe chemotherapeutic agent administered in combination with a Wntpathway inhibitor is an anti-mitotic agent, the cancer or tumor beingtreated is breast cancer or a breast tumor.

In some embodiments, an additional therapeutic agent comprises an agentsuch as a small molecule. For example, treatment can involve thecombined administration of a Wnt pathway inhibitor (e.g. an antibody) ofthe present invention with a small molecule that acts as an inhibitoragainst additional tumor-associated proteins including, but not limitedto, EGFR, ErbB2, HER2, and/or VEGF. In certain embodiments, theadditional therapeutic agent is a small molecule that inhibits a cancerstem cell pathway. In some embodiments, the additional therapeutic agentis a small molecule inhibitor of the Notch pathway. In some embodiments,the additional therapeutic agent is a small molecule inhibitor of theWnt pathway. In some embodiments, the additional therapeutic agent is asmall molecule inhibitor of the BMP pathway. In some embodiments, theadditional therapeutic agent is a small molecule that inhibits β-cateninsignaling.

In some embodiments, an additional therapeutic agent comprises abiological molecule, such as an antibody. For example, treatment caninvolve the combined administration of a Wnt pathway inhibitor (e.g. anantibody) of the present invention with other antibodies againstadditional tumor-associated proteins including, but not limited to,antibodies that bind EGFR, ErbB2, HER2, and/or VEGF. In certainembodiments, the additional therapeutic agent is an antibody that is ananti-cancer stem cell marker antibody. In some embodiments, theadditional therapeutic agent is an antibody that binds a component ofthe Notch pathway. In some embodiments, the additional therapeutic agentis an antibody that binds a component of the Wnt pathway. In certainembodiments, the additional therapeutic agent is an antibody thatinhibits a cancer stem cell pathway. In some embodiments, the additionaltherapeutic agent is an antibody inhibitor of the Notch pathway. In someembodiments, the additional therapeutic agent is an antibody inhibitorof the Wnt pathway. In some embodiments, the additional therapeuticagent is an antibody inhibitor of the BMP pathway. In some embodiments,the additional therapeutic agent is an antibody that inhibits β-cateninsignaling. In certain embodiments, the additional therapeutic agent isan antibody that is an angiogenesis inhibitor or modulator (e.g., ananti-VEGF or VEGF receptor antibody). In certain embodiments, theadditional therapeutic agent is bevacizumab (AVASTIN), trastuzumab(HERCEPTIN), panitumumab (VECTIBIX), or cetuximab (ERBITUX). Combinedadministration can include co-administration, either in a singlepharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously.

Furthermore, treatment with a Wnt pathway inhibitor described herein caninclude combination treatment with other biologic molecules, such as oneor more cytokines (e.g., lymphokines, interleukins, tumor necrosisfactors, and/or growth factors) or can be accompanied by surgicalremoval of tumors, cancer cells, or any other therapy deemed necessaryby a treating physician.

It will be appreciated that the combination of a Wnt pathway inhibitorand an additional therapeutic agent may be administered in any order orconcurrently. In some embodiments, the Wnt pathway inhibitor isadministered to subjects that have previously undergone treatment with asecond therapeutic agent. In certain other embodiments, the Wnt pathwayinhibitor and a second therapeutic agent is administered substantiallysimultaneously or concurrently. For example, a subject may be given aWnt pathway inhibitor (e.g., an antibody) while undergoing a course oftreatment with a second therapeutic agent (e.g., chemotherapy). Incertain embodiments, a Wnt pathway inhibitor is administered within 1year of the treatment with a second therapeutic agent. In certainalternative embodiments, a Wnt pathway inhibitor is administered within10, 8, 6, 4, or 2 months of any treatment with a second therapeuticagent. In certain other embodiments, a Wnt pathway inhibitor isadministered within 4, 3, 2, or 1 weeks of any treatment with a secondtherapeutic agent. In some embodiments, a Wnt pathway inhibitor isadministered within 5, 4, 3, 2, or 1 days of any treatment with a secondtherapeutic agent. It will further be appreciated that the two (or more)agents or treatments may be administered to the subject within a matterof hours or minutes (i.e., substantially simultaneously).

As is known to those of skill in the art, administration of anytherapeutic agent may lead to side effects and/or toxicities. In somecases, the side effects and/or toxicities are so severe as to precludeadministration of the particular agent at a therapeutically effectivedose. In some cases, drug therapy must be discontinued, and other agentsmay be tried. However, many agents in the same therapeutic class oftendisplay similar side effects and/or toxicities, meaning that the subjecteither has to stop therapy, or if possible, suffer from the unpleasantside effects associated with the therapeutic agent.

Side effects from therapeutic agents may include, but are not limitedto, hives, skin rashes, itching, nausea, vomiting, decreased appetite,diarrhea, chills, fever, fatigue, muscle aches and pain, headaches, lowblood pressure, high blood pressure, hypokalemia, low blood counts,bleeding, and cardiac problems.

Thus, in some embodiments, the methods described herein include using anintermittent dosing regimen, which may reduce side effects and/ortoxicities associated with administration of a Wnt pathway inhibitor. Asused herein, “intermittent dosing” refers to a dosing regimen using adosing interval of more than once a week, e.g., dosing once every 2weeks, once every 3 weeks, once every 4 weeks, etc. In some embodiments,a method for treating a subject comprises administering to the subjectan effective dose of a Wnt pathway inhibitor (e.g., an anti-FZD antibodyor a FZD soluble receptor) according to an intermittent dosing regimen.In some embodiments, the method comprises administering to the subjectan effective dose of a Wnt pathway inhibitor (e.g., an anti-FZD antibodyor a FZD soluble receptor) according to an intermittent dosing regimen,and increasing the therapeutic index of the Wnt pathway inhibitor. Insome embodiments, the intermittent dosing regimen comprisesadministering an initial dose of a Wnt pathway inhibitor to the subject,and administering subsequent doses of the Wnt pathway inhibitor aboutonce every 2 weeks. In some embodiments, the intermittent dosing regimencomprises administering an initial dose of a Wnt pathway inhibitor tothe subject, and administering subsequent doses of the Wnt pathwayinhibitor about once every 3 weeks. In some embodiments, theintermittent dosing regimen comprises administering an initial dose of aWnt pathway inhibitor to the subject, and administering subsequent dosesof the Wnt pathway inhibitor about once every 4 weeks.

In some embodiments, the subsequent doses in an intermittent dosingregimen are about the same amount or less than the initial dose. Inother embodiments, the subsequent doses are a greater amount than theinitial dose. As is known by those of skill in the art, doses used willvary depending on the clinical goals to be achieved. In someembodiments, the initial dose is about 0.25 mg/kg to about 20 mg/kg. Insome embodiments, the initial dose is about 0.25, 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg. In certainembodiments, the initial dose is about 0.5 mg/kg. In certainembodiments, the initial dose is about 1 mg/kg. In certain embodiments,the initial dose is about 2.5 mg/kg. In certain embodiments, the initialdose is about 5 mg/kg. In certain embodiments, the initial dose is about7.5 mg/kg. In certain embodiments, the initial dose is about 10 mg/kg.In certain embodiments, the initial dose is about 12.5 mg/kg. In certainembodiments, the initial dose is about 15 mg/kg. In certain embodiments,the initial dose is about 20 mg/kg. In some embodiments, the subsequentdoses are about 0.25 mg/kg to about 15 mg/kg. In certain embodiments,the subsequent doses are about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 mg/kg. In certain embodiments, the subsequent doses areabout 0.5 mg/kg. In certain embodiments, the subsequent doses are about1 mg/kg. In certain embodiments, the subsequent doses are about 2.5mg/kg. In certain embodiments, the subsequent doses are about 5 mg/kg.In some embodiments, the subsequent doses are about 7.5 mg/kg. In someembodiments, the subsequent doses are about 10 mg/kg. In someembodiments, the subsequent doses are about 12.5 mg/kg.

In some embodiments, the intermittent dosing regimen comprises: (a)administering to the subject an initial dose of a Wnt pathway inhibitorof about 2.5 mg/kg and (b) administering subsequent doses of about 2.5mg/kg once every 2 weeks. In some embodiments, the intermittent dosingregimen comprises: (a) administering to the subject an initial dose of aWnt pathway inhibitor of about 5 mg/kg and (b) administering subsequentdoses of about 5 mg/kg once every 2 weeks. In some embodiments, theintermittent dosing regimen comprises: (a) administering to the subjectan initial dose of a Wnt pathway inhibitor of about 2.5 mg/kg and (b)administering subsequent doses of about 2.5 mg/kg once every 3 weeks. Insome embodiments, the intermittent dosing regimen comprises: (a)administering to the subject an initial dose of a Wnt pathway inhibitorof about 5 mg/kg and (b) administering subsequent doses of about 5 mg/kgonce every 3 weeks. In some embodiments, the intermittent dosing regimencomprises: (a) administering to the subject an initial dose of a Wntpathway inhibitor of about 2.5 mg/kg and (b) administering subsequentdoses of about 2.5 mg/kg once every 4 weeks. In some embodiments, theintermittent dosing regimen comprises: (a) administering to the subjectan initial dose of a Wnt pathway inhibitor of about 5 mg/kg and (b)administering subsequent doses of about 5 mg/kg once every 4 weeks. Incertain embodiments, the initial dose and the maintenance doses aredifferent, for example, the initial dose is about 5 mg/kg and thesubsequent doses are about 2.5 mg/kg. In certain embodiments, anintermittent dosing regimen may comprise a loading dose, for example,the initial dose is about 20 mg/kg and the subsequent doses are about2.5 mg/kg or about 5 mg/kg administered once every 2 weeks, once every 3weeks, or once every 4 weeks.

In some embodiments of the methods described herein, a method oftreating cancer comprises administering a therapeutically effectiveamount of OMP-18R5 to a subject in need thereof at a dosage of (a) atleast about 0.5 mg/kg about every one to two weeks or (b) at least about1.0 mg/kg about every three weeks. In some embodiments, a method oftreating cancer comprises administering a therapeutically effectiveamount of OMP-18R5 to a subject in need thereof at a dosage of about 0.5mg/kg to about 1.0 mg/kg about every one to two weeks. In someembodiments, a method of treating cancer comprises administering atherapeutically effective amount of OMP-18R5 to a subject in needthereof at a dosage of about 1.0 mg/kg to about 10.0 mg/kg about everythree weeks.

Another aspect of the present invention is directed to methods forreducing toxicity of a Wnt pathway inhibitor in a human subjectcomprises administering to the subject the Wnt pathway inhibitor usingan intermittent dosing regimen. Another aspect of the present inventionis directed to methods for reducing side effects of a Wnt pathwayinhibitor in a human subject comprises administering to the subject theWnt pathway inhibitor using an intermittent dosing regimen. Anotheraspect of the present invention is directed to methods for increasingthe therapeutic index of a Wnt pathway inhibitor in a human subjectcomprises administering to the subject the Wnt pathway inhibitor usingan intermittent dosing regimen.

The choice of delivery method for the initial and subsequent doses ismade according to the ability of the subject to tolerate introduction ofthe Wnt pathway inhibitor into the body. Thus, in any of the aspectsand/or embodiments described herein, the administration of the Wntpathway inhibitor may be by intravenous injection or intravenously. Insome embodiments, the administration is by intravenous infusion. In anyof the aspects and/or embodiments described herein, the administrationof the Wnt pathway inhibitor may be by a non-intravenous route.

In certain embodiments, the treatment involves the administration of aWnt pathway inhibitor (e.g. an antibody) of the present invention incombination with radiation therapy. Treatment with a Wnt pathwayinhibitor can occur prior to, concurrently with, or subsequent toadministration of radiation therapy. Dosing schedules for such radiationtherapy can be determined by the skilled medical practitioner.

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples, which describe the useof a Wnt pathway inhibitor for treatment of cancer. It will be apparentto those skilled in the art that many modifications, both to materialsand methods, may be practiced without departing from the scope of thepresent disclosure.

EXAMPLES Example 1 Intermittent Dosing with Anti-FZD Antibody OMP-18R5in a Breast Xenograft Model and Effect on Tumor Growth

UM-PE13 breast tumor cells (20,000 cells) were injected subcutaneouslyinto 6-8 week old NOD/SCID mice. The animals were randomized into groups(n=10 per group) and treated with anti-FZD antibody OMP-18R5 incombination with paclitaxel (Taxol) and paclitaxel alone. Paclitaxel wasadministered at 10 mg/kg weekly and OMP-18R5 was administered at dosesof 5, 10, 25, or 45 mg/kg once every 3 weeks. The agents wereadministered intraperitoneally. Tumor volumes were measured on theindicated days with electronic calipers.

As shown in FIG. 1, OMP-18R5 in combination with paclitaxel administeredevery 3 weeks was efficacious in reducing PE-13 tumor growth at doses aslow as 5 mg/kg or 10 mg/kg. This tumor growth inhibition was greaterthan the growth inhibition seem with paclitaxel alone when administeredweekly. Higher doses of OMP-18R5, 25 mg/kg and 45 mg/kg, in combinationwith paclitaxel inhibited tumor growth to an even greater extent andtumor regression was observed at later time points. These resultsdemonstrate that the efficacy of anti-FZD antibody treatment incombination with a chemotherapeutic agent such as paclitaxel ismaintained with intermittent dosing regimens.

Example 2 Effect of Intermittent Dosing with Anti-FZD Antibody OMP-18R5on Bone Formation

UM-PE13 breast tumor cells (20,000 cells) were injected subcutaneouslyinto 6-8 week old NOD/SCID mice. The animals were randomized into groups(n=10 per group) and treated with anti-FZD antibody OMP-18R5 incombination with paclitaxel (Taxol) or paclitaxel alone. Paclitaxel wasadministered at 15 mg/kg once a week and OMP-18R5 was administered at 25mg/kg once every 4 weeks, once every 2 weeks or once a week. The agentswere administered intraperitoneally. Tumor volumes were measured on theindicated days with electronic calipers.

As shown in FIG. 2, OMP-18R5 in combination with paclitaxel administeredat 25 mg/kg was efficacious in reducing PE-13 tumor growth with dosingonce a week, once every 2 weeks, and once every 4 weeks. Tumor growthinhibition with OMP-18R5 in combination with paclitaxel was greater thanthe growth inhibition seen with paclitaxel alone.

At the ending of dosing on day 77, trabecular bone formation wasassessed in the OMP-18R5 treated mice as compared to mice treated withcontrol (paclitaxel alone).

Tissue sections were prepared from the tibia of control andOMP-18R5-treated mice and stained with hemotoxylin and eosin (H&E). Thelight pink staining regions highlighted by the white arrows correspondto trabecular bone.

As observed in FIG. 3, there was a reduction in bone loss with treatmentof OMP-18R5 at 25 mg/kg once every 2 weeks as compared to treatment of25 mg/kg once every week. Importantly, treatment of OMP-18R5 at 25 mg/kgevery 4 weeks appeared to have no perceptible effect on bone formation.

Example 3 Effect of Zolendronic Acid in Reducing the Effect of OMP-18R5on Bone Formation

NOD/SCID mice were randomized into groups (n=5 per group) and treatedwith anti-FZD antibody OMP-18R5 or OMP-18R5 in combination withzolendronic acid. Mice were treated with 20 mg/kg OMP-18R5 on days 1 and15 only, or 20 mg/kg OMP-18R5 on days 1 and 15 in combination with asingle IV dose of 100 ug/kg zoledronic acid on day 1. At the end ofdosing on day 29, femurs and tibias from mice treated with OMP-18R5alone were compared to femurs and tibias from mice treated with thecombination of OMP-18R5 and zoledronic acid and to mice treated with acontrol antibody.

Tissues sections of femur and tibia were prepared as described inExample 2.

As shown in FIG. 4, a single IV administration of zoledronic acid tomice treated with OMP-18R5 resulted in subchondral bone formationcomparable to mice treated with a control antibody. Additional studieshave demonstrated that co-administration of zolendronic acid does notaffect the anti-tumor efficacy of OMP-18R5. These data support thehypothesis that bisphosphonate administration may be protective againstthe catabolic effects of Wnt inhibition, providing a path to preservebone integrity and allow the benefits of targeting the Wnt pathway.

Example 4 Phase 1 Study of OMP-18R5 in Patients with Solid Tumors

The study is an open-label Phase 1 dose-escalation study of OMP-18R5 inpatients with a solid tumor for which there is no remaining standardcurative therapy and no therapy with a demonstrated survival benefit.The primary objectives of the study are to determine the safety and themaximum tolerated dose of OMP-18R5. The secondary objectives are todetermine the rate of immunogenicity, the preliminary efficacy, and thepharmacokinetics of OMP-18R5.

The patients in the initial portion of the trial were treated with adosing regimen of OMP-18R5 of 0.5 mg/kg every week (n=3) and 1.0 mg/kgevery week (n=5). One patient who received 0.5 mg/kg once a weekdeveloped fractures of their anterior ribs and lumbar spine afterreceiving study drug for approximately 100 days. As a result, in thecurrent phase of the trial (study is ongoing and patients are stillbeing enrolled) less frequent dosing is being utilized. Specifically,the dose levels are 0.5 mg/kg once every two weeks (n=3), and 1 mg/kg(n=4), 2.5 mg/kg (n=3), 5 mg/kg, and 10 mg/kg once every 3 weeks.Cohorts of 3 subjects are treated and evaluated for dose-limitingtoxicities (DLTs) through Day 28. If 0 of 3 subjects have a DLT,escalation to the next dose cohort occurs. If 1 of 3 subjectsexperiences a DLT, 3 additional subjects are treated. If 2 or moresubjects experience a DLT, no further subjects are dosed at that leveland 3 additional subjects are added to the preceding dose cohort unless6 subjects have already been treated at that dose level. Tumorassessments are performed on Day 56 and then every 56 days thereafter.Patients with stable disease or a response at Day 56 will be allowed tocontinue to receive OMP-18R5 until disease progression.

After a patient experienced a skeletal-related (bone fracture) event,samples from the first 8 patients were used to measure four boneturnover markers—bone specific alkaline phosphatase, procollagen type 1N-terminal propeptide (P1NP), osteocalcin, and collagen type 1cross-linked C-telopeptide (β-CTX). While no change during therapy wasnoted for bone specific alkaline phosphatase, P1NP, and osteocalcin, anincrease in β-CTX was noted in all 7 subjects who had at least onefollow-up value (Table 1, increased β-CTX values are underlined).

TABLE 1 Patient Tumor Type Dose (mg/kg) Day β-CTX 1 Colorectal 0.5 QWDay 0 570 2 Colorectal 0.5 QW Day 0 196 Day 28 308 Treatment Terminated217 3 Neuroendocrine 0.5 QW Day 0 219 (carcinoid) Day 28 825 Day 56 896Treatment Terminated 708 4 Leiomyosarcoma   1 QW Day 0 298 TreatmentTerminated 401 5 Breast   1 QW Day 0 229 Day 28 681 Treatment Terminated370 6 Colorectal   1 QW Day 0 162 Day 28 598 7 Colon   1 QW Day 0 144Day 28 301 8 Pancreatic   1 QW Day 0 406 Day 28 551

Thus, β-CTX appeared to be an early and sensitive biomarker of theeffect of OMP-18R5 on bone.

Based on the initial Phase 1 study results, the study protocol wasamended to include monitoring for skeletal-related side effects and/ortoxicities with DEXA bone density scans, bone scans, and measurements ofbone turnover biomarkers bone specific alkaline phosphatase, P 1NP,osteocalcin, and β-CTX. The amended protocol also included a strategyfor treatment of skeletal-related side effects and/or toxicities. Anypatient who had at least a doubling of their β-CTX level from theirscreening value or a T-score decline to less than −2.5 in the totalfemur or L1-L4 DEXA scan measurement would be administered ananti-resorptive medication, specifically the bisphosphonate zoledronicacid. The zoledronic acid will be administered intravenously at a doseof 5 mg at the time of the doubling of the β-CTX value or decline inT-score.

Table 2 shows the results (as of January 2013) from the 10 patients whowere subsequently enrolled and treated with less frequent dosing (i.e.,intermittent dosing) of OMP-18R5 (β-CTX values at least twice as high asbaseline are underlined).

TABLE 2 Patient Tumor Type Dose (mg/kg) Day β-CTX 9 Melanoma 0.5 QOW Day0 203 Day 28 195 Day 56 287 10 Neuroendocrine 0.5 QOW Day 0 306(pancreas) Day 28 286 Day 56 304 Day 84 664 Day 112 270 Day 140 288 Day168 413 Day 196 372 Day 224 377 Day 252 363 11 Colorectal 0.5 QOW Day 0721 Day 56 327 12 Neuroendocrine   1 Q3W Day 0 689 (carcinoid) Day 28846 Day 56 707 Day 84 350 Day 112 759 Day 140 526 Day 168 967 Day 196688 13 Bladder   1 Q3W Day 0 618 14 Colon   1 Q3W Day 0 471 Day 28 76015 Colon   1 Q3W Day 0 340 Day 28 469 Day 56 586 Day 84 156 16 Breast2.5 Q3W Day 0 386 Day 28 805 Day 56 345 17 Thymic 2.5 Q3W Day 0 232 Day28 309 18 Desmoid 2.5 Q3W Day 0 607 Day 28 555

Only two of these ten patients had a doubling of their β-CTX (patient 10from a value of 306 at baseline to a value of 664 at Day 84; and patient16 from a value of 386 at baseline to a value of 805 at Day 28). Thesedata suggest that less frequent dosing of OMP-18R5 at the dose levelsstudied results in fewer rises in β-CTX and less bone toxicity.According to the amended protocol, patient 10 was administered anintravenous dose of 5 mg of zoledronic acid. Following theadministration of zoledronic acid, the β-CTX value returned toapproximately baseline, a value of 270 at day 112, and remained atapproximately that level in subsequent measurements. Patient 16 alsoreceived zolendronic acid for doubling of their β-CTX level, and theirβ-CTX levels also returned to baseline after treatment. These datasuggest that zoledronic acid blocks the bone resorptive properties ofOMP-18R5, and can be used to mitigate this skeletal-related side effect.

None of the patients enrolled in the study had a significant change intheir bone mineral density (BMD) as assessed by DEXA scans (T-scores)while on treatment with OMP-18R5 (Table 3).

TABLE 3 Patient DEXA timepoint Location T-Score 1 Screening AP spineL1-L4 −1.6 Termination AP spine L1-L4 −1.9 Screening AP spine L3 −2.0Termination AP spine L3-L4 −2.1 Screening Dual femur neck left −1.8Termination Dual femur neck right −1.7 Screening Dual femur total mean−1.7 Termination Dual femur total mean −2.2 3 Screening AP spine L1-L2−0.1 Screening AP spine L1-L4 +0.2 Termination AP spine L1-L4 +0.7Termination AP spine L3-L4 +0.5 Screening Dual femur neck left −0.1Termination Dual femur neck right +0.2 Screening Dual femur total mean+1.0 Termination Dual femur total mean +0.7 5 Screening Femur −1.2Termination Femur −1.0 Screening Lumbar spine −0.6 Termination Lumbarspine −0.5 7 Screening Femur +1.2 Termination Femur +0.7 ScreeningLumbar spine +0.9 Termination Lumbar spine +0.9 9 Screening Lumbar spine−0.7 Termination Lumbar spine −0.9 Screening Hip +0.2 Termination Hip+0.2 10 Screening AP spine L1-L2 −0.9 Screening AP spine L1-L4 −0.4Screening Dual femur neck left −1.4 Screening Dual femur total mean −0.9Day 56 Lumbar spine −0.3 Day 56 Hip −0.8 11 Screening Femur +1.0Termination Hip +0.9 Screening Lumbar spine +0.9 Termination Lumbarspine +1.1 13 Screening Lumbar spine +0.1 Termination Lumbar spine +0.3Screening Hip −0.9 Termination Hip −1.2 14 Screening Lumbar spine 3.6Termination Lumbar spine 3.9 Screening Hip 2.4 Termination Hip 2.2 16Screening Lumbar spine 0.7 Termination Lumbar spine 0.8

These data suggest that osteopenic patients can be treated with OMP-18R5without a significant risk of developing a further decline in their bonemineral density. Furthermore, it confirms that β-CTX appears to be anearly and sensitive biomarker of skeletal-related side effects and/ortoxicities resulting from treatment with a Wnt pathway inhibitor.Finally, the study has shown that the skeletal-related side effects tiedto treatment with OMP-18R5 appear to be manageable and reversible.

Example 5 Phase 1 Study of OMP-54F28 in Patients with Solid Tumors

The study is an open-label Phase 1 dose-escalation study of OMP-54F28 inpatients with a solid tumor for which there is no remaining standardcurative therapy. The primary objectives of the study are to determinethe safety and the maximum tolerated dose of OMP-54F28. The secondaryobjectives are to determine the rate of immunogenicity, the preliminaryefficacy, and the pharmacokinetics of OMP-54F28.

The patients in the initial portion of the trial were treated with adosing regimen of OMP-54F28 of 0.5 mg/kg every 3 weeks (n=3) and 1.0mg/kg every 3 weeks (n=3). This study is ongoing and patients are stillbeing enrolled. Cohorts of 3 subjects are treated and evaluated fordose-limiting toxicities (DLTs) through Day 28. If 0 of 3 subjects havea DLT, escalation to the next dose cohort occurs. If 1 of 3 subjectsexperiences a DLT, 3 additional subjects are treated. If 2 or moresubjects experience a DLT, no further subjects are dosed at that leveland 3 additional subjects are added to the preceding dose cohort unless6 subjects have already been treated at that dose level. Tumorassessments are performed on Day 56 and then every 56 days thereafter.Patients with stable disease or a response at Day 56 will be allowed tocontinue to receive OMP-54F28 until disease progression.

Based on information gathered from the Phase 1 OMP-18R5 study, anypatient who has at least a doubling of their β-CTX level from theirscreening value or a T-score decline to less than −2.5 in their totalfemur or L1-L4 DEXA scan measurement will be administered zoledronicacid. The zoledronic acid will be administered intravenously at a doseof 5 mg at the time of the doubling of the β-CTX value or decline inT-score.

Table 4 shows the results (as of January 2013) from the first 6 patientswho were enrolled and treated with OMP-54F28 once every 3 weeks (β-CTXvalues at least twice as high as baseline are underlined).

TABLE 4 Patient Tumor Type Dose (mg/kg) Day β-CTX 1 Ovarian 0.5 Q3W Day0 215 Day 28 144 Day 56 119 Treatment Terminated 104 2 Colorectal 0.5Q3W Day 0 538 Day 28 604 Treatment Terminated 1122 3 Pancreatic 0.5 Q3WDay 0 497 Day 28 360 Day 56 414 Day 84 614 4 Adenocystic   1 Q3W Day 0346 Day 28 289 5 Renal cell   1 Q3W Day 0 657 Day 28 346 6 Cervical   1Q3W Day 0 262 Day 28 238

Patient 2 had a doubling of their β-CTX from a value of 538 at baselineto a value of 1122 at Day 42. This patient's disease progressed andtreatment with OMP-54F28 was stopped. Similar to results seen withOMP-18R5 treatment, these initial data suggest that treatment withOMP-54F28 at dose levels of 0.5 mg/kg and 1.0 mg/kg once every 3 weeksresults in few rises in β-CTX and less bone toxicity. These earlyresults from treatment with OMP-54F28 are further evidence that theskeletal-related side effects tied to treatment with Wnt pathwayinhibitors appear to be manageable with reasonable mitigationstrategies.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes tothe same extent as if each individual publication, patent, or patentapplication were specifically and individually indicated to be soincorporated by reference.

Following are the sequences disclosed in the application:

18R5 Heavy chain CDR1 (SEQ ID NO: 1) GFTFSHYTLS 18R5 Heavy chain CDR2(SEQ ID NO: 2) VISGDGSYTYYADSVKG 18R5 Heavy chain CDR3 (SEQ ID NO: 3)NFIKYVFAN 18R5 Light chain CDR1 (SEQ ID NO: 4) SGDNIGSFYVH18R5 Light chain CDR2 (SEQ ID NO: 5) DKSNRPSG 18R5 Light chain CDR3(SEQ ID NO: 6) QSYANTLSL18R5 Heavy chain variable region amino acid sequence (SEQ ID NO: 7)EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYTLSWVRQAPGKGLEWVSVISGDGSYTYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCARNFIKYVFANWGQGTLVTVSS18R5 Light chain variable region amino acid sequence (SEQ ID NO: 8)DIELTQPPSVSVAPGQTARISCSGDNIGSFYVHWYQQKPGQAPVLVIYDKSNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQSYANTLSLVFGGGTKLTVLG18R5 Heavy chain amino acid sequence with predicted signal sequenceunderlined (SEQ ID NO: 9)MKHLWFFLLLVAAPRWVLSEVQLVESGGGLVQPGGSLRLSCAASGFTFSHYTLSWVRQAPGKGLEWVSVISGDGSYTYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCARNFIKYVFANWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK18R5 Light chain amino acid sequence with predicted signal sequenceunderlined (SEQ ID NO: 10)MAWALLLLTLLTQGTGSWADIELTQPPSVSVAPGQTARISCSGDNIGSFYVHWYQQKPGQAPVLVIYDKSNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQSYANTLSLVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS18R5 Heavy chain amino acid sequence without predicted signal sequence(SEQ ID NO: 11)EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYTLSWVRQAPGKGLEWVSVISGDGSYTYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCARNFIKYVFANWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK18R5 Light chain amino acid sequence without predicted signal sequence(SEQ ID NO: 12)DIELTQPPSVSVAPGQTARISCSGDNIGSFYVHWYQQKPGQAPVLVIYDKSNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQSYANTLSLVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSHuman FZD1 Fri domain amino acid sequence without predicted signalsequence (SEQ ID NO: 13)QQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTHuman FZD2 Fri domain amino acid sequence without predicted signalsequence (SEQ ID NO: 14)QFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGHuman FZD3 Fri domain amino acid sequence without predicted signalsequence (SEQ ID NO: 15)HSLFSCEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVPWPEDMECSRFPDCDEPYPRLVDLHuman FZD4 Fri domain amino acid sequence without predicted signalsequence (SEQ ID NO: 16)FGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVHuman FZD5 Fri domain amino acid sequence without predicted signalsequence (SEQ ID NO: 17)ASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCMDYNRSEATTHuman FZD6 Fri domain amino acid sequence without predicted signalsequence (SEQ ID NO: 18)HSLFTCEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHTEFLGHuman FZD7 Fri domain amino acid sequence without predicted signalsequence (SEQ ID NO: 19)QPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGHuman FZD8 Fri domain amino acid sequence without predicted signalsequence (SEQ ID NO: 20)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTHuman FZD9 Fri domain amino acid sequence without predicted signalsequence (SEQ ID NO: 21)LEIGRFDPERGRGAAPCQAVEIPMCRGIGYNLTRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCTDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCMEAPENAHuman FZD10 Fri domain amino acid sequence without predicted signalsequence (SEQ ID NO: 22)ISSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPNNGHuman FZD1 amino acids 116-227 (SEQ ID NO: 23)CQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELC Human FZD2 amino acids 39-150(SEQ ID NO: 24)CQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQIC Human FZD3 amino acids 28-133(SEQ ID NO: 25)CEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVPWPEDMECSRFPDC Human FZD4 amino acids 48-161(SEQ ID NO: 26)CDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCHuman FZD5 amino acids 33-147 (SEQ ID NO: 27)CQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCHuman FZD6 amino acids 24-129 (SEQ ID NO: 28)CEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYC Human FZD7 amino acids 49-160(SEQ ID NO: 28)CQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEIC Human FZD8 amino acids 35-148(SEQ ID NO: 30)CQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCHuman FZD9 amino acids 39-152 (SEQ ID NO: 31)CQAVEIPMCRGIGYNLTRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCTDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCHuman FZD10 amino acids 34-147 (SEQ ID NO: 32)CQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCHuman FZD8 Fri domain amino acid sequence without predicted signalsequence (variant) (SEQ ID NO: 33)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLHuman IgG₁ Fc region (SEQ ID NO: 34)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG₁ Fc region (variant) (SEQ ID NO: 35)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG₁ Fc region (SEQ ID NO: 36)KSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG₁ Fc region (SEQ ID NO: 37)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG₂ Fc region (SEQ ID NO: 38)CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFZD8-Fc variant 54F03 amino acid sequence (without predicted signalsequence) (SEQ ID NO: 39)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTGRADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFZD8-Fc variant 54F16, 54F17, 54F18, 54F23, 54F25, 54F27, 54F29, 54F31,and 54F34 amino acid sequence (without predicted signal sequence)(SEQ ID NO: 40)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FZD8-Fc variant 54F19, 54F20, 54F24, 54F26, 54F28, 54F30, 54F32,54F34 and 54F35 amino acid sequence (without predicted signal sequence)(SEQ ID NO: 41)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FZD8-Fc variant 54F03 amino acid sequence with signal sequence(SEQ ID NO: 42)MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTGRADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFZD8-Fc variant 54F16 amino acid sequence with signal sequence(SEQ ID NO: 43)MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFZD8-Fc variant 54F26 with signal sequence (SEQ ID NO: 44)MEWGYLLEVTSLLAALFLLQRSPIVHAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFZD8-Fc variant 54F28 with signal sequence (SEQ ID NO: 45)MEWGYLLEVTSLLAALLLLQRSPFVHAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman Wnt1 C-terminal cysteine rich domain (aa 288-370) (SEQ ID NO: 46)DLVYFEKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTFHWCCHVSCRNCTHTRVLHECL Human Wnt2 C-terminal cysteine rich domain (aa 267-360)(SEQ ID NO: 47)DLVYFENSPDYCIRDREAGSLGTAGRVCNLTSRGMDSCEVMCCGRGYDTSHVTRMTKCGCKFHWCCAVRCQDCLEALDVHTCKAPKNADWTTATHuman Wnt2b C-terminal cysteine rich domain (aa 298-391) (SEQ ID NO: 48)DLVYFDNSPDYCVLDKAAGSLGTAGRVCSKTSKGTDGCEIMCCGRGYDTTRVTRVTQCECKFHWCCAVRCKECRNTVDVHTCKAPKKAEWLDQTHuman Wnt3 C-terminal cysteine rich domain (aa 273-355) (SEQ ID NO: 49)DLVYYENSPNFCEPNPETGSFGTRDRTCNVTSHGIDGCDLLCCGRGHNTRTEKRKEKCHCIFHWCCYVSCQECIRIYDVHTCK Human Wnt3a C-terminal cysteine rich domain (aa 270-352)(SEQ ID NO: 50)DLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCK Human Wnt7a C-terminal cysteine rich domain (aa 267-359)(SEQ ID NO: 51)DLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK Human Wnt7b C-terminal cysteine rich domain (aa 267-349)(SEQ ID NO: 52)DLVYIEKSPNYCEEDAATGSVGTQGRLCNRTSPGADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNTCSERTEVFTCK Human Wnt8a C-terminal cysteine rich domain (aa 248-355)(SEQ ID NO: 53)ELIFLEESPDYCTCNSSLGIYGTEGRECLQNSHNTSRWERRSCGRLCTECGLQVEERKTEVISSCNCKFQWCCTVKCDQCRHVVSKYYCARSPGSAQSLGRVWFGVYIHuman Wnt8b C-terminal cysteine rich domain (aa 245-351) (SEQ ID NO: 54)ELVHLEDSPDYCLENKTLGLLGTEGRECLRRGRALGRWELRSCRRLCGDCGLAVEERRAETVSSCNCKFHWCCAVRCEQCRRRVTKYFCSRAERPRGGAAHKPGRKPHuman Wnt10a C-terminal cysteine rich domain (aa 335-417)(SEQ ID NO: 55)DLVYFEKSPDFCEREPRLDSAGTVGRLCNKSSAGSDGCGSMCCGRGHNILRQTRSERCHCRFHWCCFVVCEECRITEWVSVCK Human Wnt10b C-terminal cysteine rich domain (aa 307-389)(SEQ ID NO: 56)ELVYFEKSPDFCERDPTMGSPGTRGRACNKTSRLLDGCGSLCCGRGHNVLRQTRVERCHCRFHWCCYVLCDECKVTEWVNVCK Linker (SEQ ID NO: 57) ESGGGGVT Linker (SEQ ID NO: 58)LESGGGGVT Linker (SEQ ID NO: 59) GRAQVT Linker (SEQ ID NO: 60) WRAQVTLinker (SEQ ID NO: 61) ARGRAQVT

1-84. (canceled)
 85. A method for reducing a skeletal-related side effect and/or toxicity in a subject receiving treatment with a Wnt pathway inhibitor, comprising: (a) administering a therapeutically effective amount of a Wnt pathway inhibitor to a subject in need thereof; (b) determining the level of collagen type 1 cross-linked C-telopeptide (β-CTX) in a sample from the subject after the administration of the Wnt pathway inhibitor; and (c) administering to the subject a therapeutically effective amount of a bisphosphonate if the level of β-CTX in the sample is higher than a predetermined level of the β-CTX; and wherein the Wnt pathway inhibitor is: (i) an antibody that specifically binds at least one frizzled (FZD) protein and comprises a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), and a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:6); or (ii) a soluble receptor comprising a Fri domain of human FZD8 and a human Fc region.
 86. The method of claim 85, wherein the Wnt pathway inhibitor is an antibody that specifically binds at least one frizzled (FZD) protein and comprises a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), and a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:6).
 87. The method of claim 85, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO:7 and a light chain variable region comprising SEQ ID NO:8.
 88. The method of claim 86, wherein the antibody is a monoclonal antibody, a recombinant antibody, a chimeric antibody, a bispecific antibody, a humanized antibody, a human antibody, or a antibody fragment comprising an antigen-binding site.
 89. The method of claim 86, wherein the antibody is OMP-18R5.
 90. The method of claim 85, wherein the Wnt pathway inhibitor is a soluble receptor comprising a Fri domain of human FZD8 and a human Fc region.
 91. The method of claim 90, wherein the soluble receptor comprises SEQ ID NO:41.
 92. The method of claim 90, wherein the soluble receptor is OMP-54F28.
 93. The method of claim 85, wherein the sample is blood, serum, or plasma.
 94. The method of claim 85, wherein the predetermined level of β-CTX is determined at an earlier timepoint, at an initial screening, and/or prior to treatment.
 95. The method of claim 85, wherein the level of β-CTX in the sample is at least two-fold higher than the predetermined level of the β-CTX.
 96. The method of claim 85, wherein the skeletal-related side effect and/or toxicity is an increased risk of bone fracture, osteopenia, or osteoporosis.
 97. The method of claim 85, wherein the bisphosphonate is zoledronic acid.
 98. The method of claim 85, wherein the subject has cancer.
 99. The method of claim 85, wherein the subject is treated with the Wnt pathway inhibitor in combination with one or more additional therapeutic agents.
 100. A method of selecting a subject for treatment with a Wnt pathway inhibitor, comprising: (a) determining the level of β-CTX in a sample from the subject; (b) selecting the subject for treatment with the Wnt pathway inhibitor if the level of β-CTX is below a predetermined level; and (c) administering a therapeutically effective dose of the Wnt pathway inhibitor to the subject; wherein the Wnt pathway inhibitor is: (i) an antibody that specifically binds at least one frizzled (FZD) protein and comprises a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), and a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:6); or (ii) a soluble receptor comprising a Fri domain of human FZD8 and a human Fc region.
 101. The method of claim 100, wherein the sample is blood, serum, or plasma.
 102. The method of claim 100, wherein the pre-determined level of β-CTX sample is a level of β-CTX determined at an initial screening.
 103. The method of claim 100, wherein the predetermined level of β-CTX is 1000 pg/ml.
 104. A method of preventing or attenuating the development of a skeletal-related side effect and/or toxicity in a subject in need of treatment with a Wnt pathway inhibitor, comprising: (a) determining the level of β-CTX in a sample from the subject prior to treatment with the Wnt pathway inhibitor; (b) administering to the subject a therapeutically effective amount of a bisphosphonate if the level of β-CTX in the sample is higher than a predetermined level of β-CTX; and (c) administering to the subject the Wnt pathway inhibitor; wherein steps (b) and (c) are performed in any order after step (a); and wherein the Wnt pathway inhibitor is: (i) an antibody that specifically binds at least one frizzled (FZD) protein and comprises a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), and a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:6); or (ii) a soluble receptor comprising a Fri domain of human FZD8 and a human Fc region.
 105. A method of preventing or attenuating the development of a skeletal-related side effect and/or toxicity in a subject receiving treatment with a Wnt pathway inhibitor, comprising administering to the subject a therapeutically effective amount of a bisphosphonate; wherein the Wnt pathway inhibitor is: (i) an antibody that specifically binds at least one frizzled (FZD) protein and comprises a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), and a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:6); or (ii) a soluble receptor comprising a Fri domain of human FZD8 and a human Fc region.
 106. A method of treating cancer in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of a Wnt pathway inhibitor; and (b) determining the level of f3-CTX in a sample from the subject; wherein the Wnt pathway inhibitor is: (i) an antibody that specifically binds at least one frizzled (FZD) protein and comprises a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), and a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:6); or (ii) a soluble receptor comprising a Fri domain of human FZD8 and a human Fc region. 